Mechanical simulations in tissue engineering

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Effect of mechanical stimulus on cardiac, bone and cartilage tissue

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Mechanical simulations in tissue engineering

  1. 1. EPFL, Tissue EngineeringSemester PaperMechanical Stimulation in Tissue EngineeringAuthors: Anna Cyganowski, Nadia Vertti, Saurabh KhemkaProfessor : Dr. Peter FreySpring Semester 2012
  2. 2. Contents1 Introduction 11.1 The promise of tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Why mechanical stimulation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Current Developments 22.1 Cardiac Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2Cardiac Tissue Engineering Principles . . . . . . . . . . . . . . . . . . . . . . . 22.1.2 Present State of the Art . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Effects of mechanical stretching . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Effects of mechanical pulsing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Effects of diminished mechanical pulsing . . . . . . . . . . . . . . . . . . . . . . 32.2 Bone Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Bone Tissue Engineering Principles . . . . . . . . . . . . . . . . . . . . . . . . . 52.2.2 Present State of the Art . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Mechanical stimulation using perfusion based bioreactor . . . . . . . . . . . . . 6Mechanical stimulation using direct mechanical strain . . . . . . . . . . . . . . 72.3 Cartilage Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82.3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Cartilage Tissue Engineering Principles . . . . . . . . . . . . . . . . . . . . . . 92.3.2 Present State of the Art . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9System for studying chondrocytes . . . . . . . . . . . . . . . . . . . . . . . . . 9Mechanical input . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Measurement of cell response . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112.3.3 Example of cell engineering mechanotransduction study . . . . . . . . . . . . . 113 Future Developments: Microfluidics and Microtechnology 134 Conclusions 14ii
  3. 3. 1 Introduction1.1 The promise of tissue engineeringTissue engineering is the final frontier in the field of regenerative medicine. Ever since a group ofMIT researchers presented the infamous Vacanti mouse in 1995, scientists, engineers, clinicians, andthe general public alike have eagerly anticipated the technological advancements that promised to healinjuries and cure diseases. Instead of relying on an insufficient supply of organ donations or syntheticmaterials that integrated poorly with the body’s immune system, patients found hope in the idea thattheir own biological cells could be grown into the tissues or organs they needed to survive.Yet nearly thirty years after the term “tissue engineering” was first used, the technologies thatseemed promising in the lab have yet to make it to the clinic for general use. And, the few technologiesthat have been tried, such as synthetic skin for severe burn patients, have severe limits, such asthe lack of sweat glands and hair follicles for cooling the body. Because tissue engineering spans asignificant number of unknowns between biology, medicine, and engineering, this complex field needscontributions from diverse fields in order to succeed.1.2 Why mechanical stimulation?Traditionally, biologists cultured cells by seeding them into an environment that enabled them sur-vive, with adequate nutrients and temperature. However, in order for cells to develop into viable tissuefor transplantation, they must do more than simply sustain life. Tissue engineers have discovered thatreplicating the in vivo environment to which a particular tissue or organ would naturally be exposedimproves the differentiation, organization, and three-dimensional development of cells. Mechanicalstimulation is one factor that is present during cell development, whether during the early fetal stagesor later during the growth, maintenance, and repair of an organ. This is because of a phenomenonknown as mechanotransduction, a process where cells convert mechanical stimuli into electrical orchemical signals through the cell membrane ion channels. These signals regulate cell processes, suchas the synthesis of growth hormones, which affect cell development into tissue. For these reasons, thestudy of mechanical stimulation is key to the advancement of tissue engineered therapies.Though there is a vast amount of research on the role of mechanical stimulation in tissue engineering,this report will focus specifically on three tissue types: cardiac, bone, and cartilage.1
  4. 4. 2 Current Developments2.1 Cardiac Tissue2.1.1 IntroductionIn 2008, 1 in 4 deaths in the United States were caused by heart disease, making it the leading causeof mortality in the country. In addition, over 7 million Americans have suffered from heart attacks, astaggering statistic that is predicted to worsen as the general population ages [21]. After myocardialischemia, the narrowing of the artery causes oxygen depletion and massive localized cell death, whichresults in fibrotic scarring and impaired cardiac function. At this point, the heart is unable to repairitself for two reasons. First, cardiomyocytes, the cells that compose the heart, are limited in theirregeneration potential [3]. Second, the number of myoblasts, the muscle cell type that can divideand differentiate to form new cells, in cardiac tissue is low [2]. Currently, the most successful clinicaltreatment of a failing heart is organ transplantation, but because of a shortage in organ donations aswell as the potential risks of immune reactions, new treatments must be developed.Cardiac Tissue Engineering PrinciplesThere are several potential methods of myocardial repair currently being studied. One is the directtransplantation of healthy cells into the damaged areas. To replace necrotic tissue and reduce scarring,several cell types have been tested, including fetal cardiomyocytes and skeletal myoblasts [12]. Theidea is to graft these cells on top of the wounded areas of the heart to encourage repair. Alternatively,stem cells from the bone marrow or peripheral blood have been injected into the wounded site toinduce cardiac muscle and vascular tissue growth [1]. However, none of these methods have succeededin producing enough myocardial fibers to prevent cell death after engraftment. Instead, the engraftedmyoblasts fail to differentiate and integrate with the cardiac host, which prevents electromechanicalcoupling [12]. This lack of synthesis increases the patients risk for more heart problems in the future,such as arrhythmias.Tissue engineering has the potential to provide better solutions to this problem. By engineeringcardiac tissue from scaffolds or biomaterial gels seeded with cells, an engineer can better controlthe parameters of the engineered constructs, such as shape, size, development, and function [12].Ultimately, one goal of cardiac tissue engineering is to develop a cell sheet patch that can be graftedto the heart to stimulate regeneration. However, the main challenge is creating a muscle that isrobust while reducing the risk for necrosis after transplantation. Zimmerman et. al. proposed thata successful cardiac tissue engineered construct should meet the following five criteria: contractility,electrophysiological stability, flexibility and mechanical robustness, vascularization or the ability to bequickly vascularized in vivo, and non-immunogenicity [23]. To date, engineered tissues have not yetmet all of these criteria.Among the techniques used to improve the differentiation of cardiac tissue, many have the samegoal in mind: to mimic the complex environment of the developing heart in vivo. Though thereare many factors that contribute to this, such as biochemical signaling, the mechanical stimulationexperienced by cardiac tissue is perhaps the most obvious. Pumping over 40 million times a yearat 60-100 beats per minute [23], the heart experiences a significant amount of stresses and strains.Studying the boundary conditions of this mechanical stimulation as experienced by fetal tissue wouldnot only expand scientific knowledge in the field of developmental biology, but would also give tissueengineers the tools they need to replicate this phenomenon in vitro.2
  5. 5. 2.1.2 Present State of the ArtEffects of mechanical stretchingMany researchers have incorporated the use of mechanical stimulation for cardiac tissue engineering,primarily in bioreactor environments. Some of the earliest and most cited work has been done byZimmerman et. al. at the University of Hamburg in Germany. This group created biomaterialconstructs of a combination of collagen type I, extracellular matrix proteins, and freshly isolated heartcells from neonatal rats and embryonic chickens. The advantage of using this mixture is that it canbe casted into molds of various shapes, which allows for more experimental structures, ranging fromlattice to circular geometries [4]. After culturing for 5-7 days, the developing tissue solidified andbegan to contract. To further enhance development, the construct continued to culture with addedcyclic mechanical stretch for 5-10 more days.The group found significant evidence to suggest that mechanical stimulation improved the develop-ment of the engineered heart tissue. First, the morphology of the stretched tissue was more advanced,with denser regions of more developed and longitudinally oriented rod-shaped cells. The cross-sectionaldiameter of the stretched cells were 41% greater than those without stretching [4]. The contractileforces of the cells after mechanical stimulation were between 2-4 times higher than the control cells,both at rest and during stimulation with calcium. Fig. 2.1 shows the experimental setup, includingthe culture dish with engineered heart tissue structures before stretching, the motorized stretchingdevice, and the schematic representation of the phasic stretch used at 1.5 Hz. While this work wasuseful in showing that mechanical stimulation is beneficial to the development of engineered tissue,the results have their limits. The engineered tissue constructs in this case are only 0.4 millimeters,which is too thin to use in grafting applications.Effects of mechanical pulsingAnother method of introducing mechanical stimulation to tissue culture is through the use of biore-actors. Simon Hoerstrup et. al developed an in vitro pulse duplicator system to focus on a differentaspect of cardiac tissue engineering. Instead of developing cardiac tissue, the goal of this group wasto engineer heart valves in vitro as a substitute for the mechanical heart valve devices used in theclinic today. Though heart valve devices give patients a practical alternative to organ transplants – anoption that is unavailable for those who have cardiac tissue damage – these mechanical devices oftenfail in 30-35% of patients within 10 years [8] . Some problems caused by mechanical valves include anincreased risk of thromboembolism and tissue deterioration. Especially in young and growing children,the durability and size limitations of mechanical valves are not ideal. Previous tissue engineered heartvalves are also structurally immature and mechanically weak. To improve the development of heartvalves in vitro, these constructs were cultured in a bioreactor for 14 days, with flow and pressureconditions gradually increasing. The flow ranged from 125 mL/min to 750 mL/min and the pressureincreased from 30 mm Hg to 55 mm Hg. Afterwards, cardiopulmonary bypass was used to implantthe engineered constructs into lambs and later removed after 1 day and 4, 6, 8, 16, and 20 weeks [8].The results of this experiment were encouraging. After 20 weeks of implantation in the lambs,the heart valve tissue constructs were found to be mechanically comparable to native tissue. Theextracellular matrix and DNA levels were also similar to native tissue at that point in development.However, there is a great need for more future work before this technology can be transferred to theclinic. Though the engineered heart valve was successful in vivo at 20 weeks, the longer term fate isunknown. In addition, since the number of implants in this experiment was small, more tests needto be done to increase statistical significance. Other improvements to this work include creating ascaffold that is more accurate in shape, finding the optimal cell source for seeding, and optimizing thepressure loading conditions in the bioreactor to simulate the biological environment.Effects of diminished mechanical pulsingA more improved bioreactor design has been implemented by Yuji Narita et. al., which uses a pulseduplicating system that is capable of softening the usual peaky pressure waveforms seen in mechanical3
  6. 6. Figure 2.1: Setup used by Zimmerman et. al. to stretch engineered heart tissue constructs [4]stimulation [14]. Fig. 2.2 shows the general structure of this bioreactor, which includes a balloonchamber with a mechanical valve and a compliance chamber that softens the peaks of mechanicalstimulation. Perviously, similar bioreactors for mechanical stimulation included a compressed air res-pirator with a peristaltic pump, and couldn’t handle various types and shapes of scaffold materials.Because of the balloon chamber, the tissue constructs could be exposed to both shear and stretchstresses, and a wide range of pulsatile flows. This also means that the pressure profile resembledmore closely the kind seen in vivo during natural development. The results of this experiment clearlydemonstrated an improvement: the cell counts, protein content, and pproteo-glycan-glycosamino gly-can levels of tissue constructs grown in this bioreactor was higher than those in both the static andtraditional ”peaky pressure” stimulation chambers [14].2.2 Bone Tissue2.2.1 IntroductionBone, a tissue with remarkable rigidity, provides structure to the body. Bone not only supportsand protects the internal part of the body, but also produces blood cells and stores minerals andgrowth factors. Apart from traumatic accidental injuries, there are several other common bone defectsthat need treatment, such as osteomyelitis (bone or bone marrow infection), osteogenesis imperfecta(genetic bone disorder), and osteoporosis (brittle bone disease).Osteoarthritis (degenerative joint disease) alone is found to affect 26.9 million US adults as of 2005with more than 780,000 total joint replacements in 2006. In 1997, total expenditure for knee andjoint replacement was 7.9 billion dollars. Currently, the standard procedure for critical-sized bone4
  7. 7. Figure 2.2: Pulse duplicating bioreactor with the ability to avoid peaky pressure waveforms [14]defects is autogenous grafting, in which bone from another site is taken and grafted. However, highercomplications, pain at the donor site, increased risk of infections, and restricted availability limitsits application in traumatic bone injuries. Alternatively, the use of bone tissue from other humans,known as allografting, is being employed, but the risk of infection transfer, host immune response, andlimited availability are associated concerns. Xenografting, the grafting of bone from a non-human, isconsidered to be unsuitable due to increased risk of disease, virus transmission, host rejection, andimmunogenicity.The aforementioned problems associated with current procedures limits the supply of the graft whilethe demand increases year by year, forcing scientists, engineers, and clinicians to look for alternatives.Tissue engineering seems to be a promising in producing new bone tissue by utilizing the body’snatural response to tissue damage.Bone Tissue Engineering PrinciplesTissue engineering aims to built the organs in vivo, ready to be transplanted to patients in need.To grow living tissue in vitro, cultured cells are seeded on bioactive degradable scaffolds that pro-vide the physical and chemical cues to guide their differentiation and assembly into three-dimensional(3D) tissues. An ideal bone scaffold should present a physiochemical biomimetic environment, pro-vide temporary mechanical support to the affected area, contain a porous structure allowing bone cellmigration and vascularization, and deliver drugs in a controlled manner to enhance rapid healing. [16]In a scaffold, cells are seeded and allowed to proliferate. Osteoblasts have been widely used as thesecells produce bone extracellular matrix (ECM) in the scaffold; however, their ability to proliferate issignificantly less than their precursor, mesenchymal stromal cells (MSCs). MSCs are mostly isolatedfrom bone marrow aspirates and have the ability to differentiate into diverse mesenchymal lineages,including osteoblasts, chondrocytes, adipocytes, and myocytes. [15] One major problem with thisapproach of in vitro culturing of cells in scaffolds to produce bone grafts is the long time required tocreate the scaffold, which forces the patient to wait. In order to enhance growth, various techniquesare being employed including the use of growth factors and bioreactors that provide mechanical stim-ulation. This section of the report will focus on the effect of mechanical transduction on bone tissueengineering.The inferior mechanical properties of tissue engineered constructs has driven researchers to parsethe role of mechanotransduction. This process converts mechanical stimuli into biochemical signals,leading to the adaption of bone to the mechanical loading. One of the most important mechanicalstimuli is the shear stress produced by the blood flow in bone porous space. For humans, the levelof shear stress is in the range of 0.8-3 Pa. Moreover, the muscle contraction and body movementsresult in continuous exposure of bone to mechanical stimuli by changing hydrostatic pressure, directcell strain, fluid flow-induced shear stress, and electric fields.[16] It has been demonstrated that fluidinduced shear stress enhances the intracellular Ca2+ release in vascular endothelial cells, activating5
  8. 8. Figure 2.3: apparatus used for Diagram of mechanical strain (A) and fluid flow (B) experiments.[20]other proteins of signaling pathways. Moreover, it has been suggested that fluid flow up regulates theexpression of many signaling molecules, paracrine factors (example- prostaglandin E2) and mRNAof bone matrix proteins (e.g., osteopontin). Different growth factors, such as the insulin/like BMPs,have also been found to play role in mechanical stimulation. In recent years, various bioreactors havebeen designed to mimic the in vivo environment by facilitating the proper nutrient supply across the3D scaffold, cell growth, and mechanical stimulation. In the following section, we explore the presentstate-of-art in bone tissue engineering and role of mechanical stimulation.2.2.2 Present State of the ArtMechanical stimulation using perfusion based bioreactorIn early 1997, R. Smalt and colleagues compared the ability of direct mechanical strain and fluid flowinduced shear stress to stimulate the bone cell growth. Rat calvarial osteoblast cells were extractedand suspended in adequate buffer. For the mechanical strain testing, the cells were added to wells ona tissue culture-treated strip of polystyrene film followed by the attachment to metals bars capable ofmoving relative to each other by the electromagnetic force (Fig.2.3). While in flow experiments, glassslides were mounted on a parallel plate flow chamber, and HEPES-buffer with 0.1 % bovine serumalbumin was allowed to flow in controlled manner. Shear stress of the range 0-80 dyn/cm2 was applied(Fig.2.3). They observed that although the bone cells were unresponsive to mechanical strain up to5,000 µ-strain, both primary cultures containing osteoblastic cells and osteoblastic cell lines respondedto even low levels of wall-shear stress, resulting in higher production of PEG2. It was shown thatfluid flow shear stress in the range of 8-30 dyn/cm2 is more likely to be the stimulus acting on bonecells in vivo. [20]Although the above mentioned experiment demonstrated the ability of flow induced shear stressin stimulating cell growth, the experiment was limited by the use of monolayer culturing. This isquite different from the environment in vivo, which is 3-dimensional. Perfusion bioreactors have beendesigned to achieve the flow induced shear stress with low rate of perfusion (e.g., 0.01-3 mL/min)to provide adequate diffusion of nutrients through 3D scaffolds and necessary shear stress for cellproliferation. By mimicking the physiological loading of bones, which is cyclic in nature (e.g. thedirection of fluid flow in stance phase of gait is different from the swing phase of the gate), Vance etal. in 2005 used perfusion bioreactors with oscillating fluid flow. They used the mouse osteoblastic6
  9. 9. cell line MC3T3 cultured in standard MC3T3 medium, which were statically seeded onto the calciumphosphate scaffolds having the porous structure similar to trabecular bone. The bioreactor in which theseeded scaffold were loaded comprised a syringe pump providing the constant low perfusion flow ratewith a linear actuator to generates a larger magnitude flow (mechanical stimulus) and an oscillatoryflow profile apart from other parts of bioreactor (Fig. 2.4). They observed that PGE2 levels increasedFigure 2.4: (A) Bioreactor system: a linear actuator and syringe pump are used to drive culturemedium through the bioreactor chambers. Flow rates and profiles are monitored with aflow probe. (B) Bioreactor flow chamber viewed in longitudinal section. A single calciumphosphate scaffold seeded with cells. [22]by 2.5-fold and 4.5 folds compared from static controls, to an average fluid-induced shear stress of0.0007 Pa from perfusion flow and 30 min of oscillating flow at 1 Hz, producing a shear stress ofapproximately 1.2 Pa, interposed on the same perfusion flow respectively. [22] These findings clearlydemonstrate the role of mechanical stimulation in enhancing the proliferation of osteoblastic cells.Recent advances include use of decellularized bovine cancellous bone (cylindric, 4X4 mm) as ascaffold seeded with human MSCs to mimic biological topography in an novel perfusion bioreactorcapable of cultivating six tissue constructs simultaneously. By increasing the flow rate from 100 mm/sto 400 mm/s increment in uniform cell distribution, Collagen growth, osteopontin, bone sialoprotein(BSP2) was observed. [6]Similarly in 2008, Jagodzinski et al. observed the increase in cell proliferationand osteocalcin (OC) level increment in above mentioned scaffold of size 4x20mm using a custom madeperfusion based reactor with cyclic compression. [10]. These finding clearly demonstrate the role ofmechanical stimulation in enhancing the proliferation of osteoblastic cells, different growth factors andsignaling molecules.Mechanical stimulation using direct mechanical strainIn 1892, Julius Wolff depicted the role of mechanical loading in bone remodeling. Although theexact mechanisms are still unknown, Wolff found that loading the bone with various strains affectedbone loss, growth, and maintenance. Strains below 500 µ-strain resulted in bone loss, while loadingup to 1000 µ-strain maintained the original bone geometry and mass. Meanwhile, strains between1000 - 4000µ-strain increased new bone formation progressively. [17]Various type of direct mechanical strain include stretching, bending, contraction or compression,and few of them will be discussed below. The use of four point bending (Fig. 2.5) in the study7
  10. 10. of human bone marrow stomal cells (hBMSCs) seeded in 3-D partially demineralized bone scaffoldhas been shown to increase osteogenic differentiation. Increased levels of alkaline phosphatase (ALP)activity (around 82–90 %), mineralized matrix production, and gene expression of ALP (by 218-257 %)and OP was also observed. The bioreactor utilized the tensile strain in the range of 44–2151 µ-strainand compressive strain of -51 to -2342 µ-strain. In this bioreactor, the force of the linear actuator istransmitted to four-point bending of the construct via the transduction beam during loading period.[13] Similarly in 2004, Ignatius et al. reported the effect of cyclic uniaxial mechanical strain (Fig.2.5) of 10,000 µ-strain on the human osteoblastic precursor cell line(hFOB1.19) in a type I collagenscaffold. They observed the high proliferation of the osteoblastic cells along with higher gene expressionof H4 (a gene related to proliferation), osteoblastic differentiation and matrix production during thecultivation period of 3 weeks. Further, the growth of ECMs were found in the direction of mechanicalstrain applied. [9] Although the existing bioreactor systems for direct mechanical stimulation haveFigure 2.5: (A) illustrates the four-point bending method. On the left, the scaffold is shown in thepassive state. On the right, mechanical loading is applied. (B) demonstrates the principleof uniaxial cyclic stretching, which is applied in elastic silicon dishes. Cells are embed-ded in three-dimensional collagen type I matrices (dark gray). (C) represents a uniaxialmechanical loading device. A plunger is pushed in a cyclic manner on the scaffold.shown beneficial effects on proliferation, osteogenic differentiation, and matrix formation, the use ofbio-mechanically unstable Collagen I gels as matrices for mechanical strain-based cellular stimulationis disadvantageous where just a certain stability is required for the initial implantation and no furtherstability is needed. [17]2.3 Cartilage Tissue2.3.1 IntroductionCartilage is a type of connective tissue in the body. It is made of cells called chondrocytes em-bedded in a matrix, and strengthened with fibers of collagen, an abundant ground substance rich inproteoglycan and sometimes elastin. It is an important tissue because of its role as a template for theosseous skeleton, a regulator of skeletal growth, the articular covering for bone ends. There are threedifferent types: hyaline cartilage, elastic cartilage, and fibrocartilage. Cartilage is avascular, meaningthat it is not supplied by blood vessels; instead, nutrients diffuse through the matrix, helped by thepumping action generated by compression of the articular cartilage or flexion of the elastic cartilage.Cartilage is usually flexible, depending on the type. Some of the bodily structures that are composed8
  11. 11. of cartilage include the ears, nose, ribcage, and intervertebral discs.According to the Department of Bioengineering of Rice University in the United States, the po-tential benefits of cartilage regeneration and replacement therapies are enormous. Tens of millions ofAmericans suffer from acute trauma to musculoskeletal tissues as well as various degenerative cartilageconditions. Currently, patients have little recourse beyond surgical techniques to ameliorate the symp-toms of disease or restore some function to damaged tissue, and cannot otherwise address the issueof healing articular cartilage. It is also important to mention the research around the consequence ofthe application of mechanical stimuli to the mesenchymal stem cells to compromise into chondrocytesthat is carried on around the world by diverse institutions. Much interest is put into the cartilagetissue development.Cartilage Tissue Engineering PrinciplesIn the tissue engineering domain, cartilage can be viewed as a biological tissue in which cells within adense extracellular matrix (ECM) are presented with a complex combination of physical forces, flows,and biological signaling factors. From the mechanical point of view, the articular cartilage (hyalineand fibrocartilage type) is subjected to a range of static and dynamical mechanical loads because ofits presence in human synovial joints. These loads can occur at a high frequency (short-duration) andat long term, even static conditions, within the physiological range. These forces can be applied in acompressive, tensile and shear way. The values of stress that cartilage receives can even reach 10 to20 MPa in some physical activities[7].In order to understand how chondrocytes form a tissue with the needed material properties, as wellas the ECM molecular structure for performing its physiological function in the human body, theirmechanisms must be understood first. This is the feedback process between physical stimuli that thecells receive and their responses (both by multiple regulatory pathways) at the molecular, cellular andtissue levels. As a consequence of the mechanical stimuli in their microenvironments, chondrocytescan synthetize, assemble, and degrade proteoglycans (PGs), collagens, glycoproteins and other matrixmolecules. One important area of study is determining how these mechanical inputs affect developingcartilage [19].2.3.2 Present State of the ArtSystem for studying chondrocytesSince the study of the mechanism by which chondrocyte respond to mechanical stimuli is difficultin vivo, there has been a development of in vitro models. These in vitro models have changed throughthe years of cell cultivation history. Three dimensional chondrocyte/gel culture systems are impor-tant nowadays because they can preserve or emulate native tissue structure and enable quantitivecorrelations between mechanical and biological parameters. Also as a consequence of delimitation ofspace, cell matrix interactions and chondrocyte gene expression can be preserved in these systems.The comprehension of the relation ECM-Chondrocytes is crucial for the tissue engineering of cartilage.Some of the hydrogels utilized by researchers for doing so are agarose, alginate and polyglycolic acid(PGA). In these substrates cells can be cultivated and then scientists look at chondrocyte phenotypicexpression, proliferation, and accumulation of a PG-rich ECM during long periods of time[7].Mechanical inputResearchers have studied the effects of various types of mechanical stimulation on cartilage cells,including applied mechanical compression (load or displacement control), hydrostatic pressure, physic-ochemical stimuli (pH and osmolarity), and electrical currents. This is because the mechanical inputsthat the cartilage receives is a combinations of mechanical stresses simultaneously during joint motionon an intermittent basis. And among these types of stimuli, there have also been a large number ofexperiments with different ranges of stimuli inputs over a broad variety of frequencies to the cells in-volved. In general, it has been found that the metabolic response to compression in vitro shows similar9
  12. 12. trends to those seen in animal studies. These suggest that static compression would significantly in-hibit the synthesis of PGs and proteins, whereas dynamic compression can markedly stimulate matrixproduction, depending on the amplitude and frequency[19].How is it possible to apply mechanical stimulation? Experiments have shown that compression ofcartilage causes deformation of cells and matrix, while hydrostatic pressure gradients create intersti-tial fluid flow. Fluid convection and separation of counterions from the fixed charge groups of theproteoglycan constituents gives rise to electrical streaming potentials and currents. In contrast, tissueshear deformation of a poroelastic tissue does not induce volumetric changes, intratissue fluid flow,or pressure gradients. All these effects of the mechanical stimuli can be modeled thanks to the devel-opment of computational simulations, which give researchers a framework for what to expect whenthey apply the mechanical stimulation to cells. The different types of mechanical stimuli are shownin figure 2.6.Figure 2.6: Schematic overview of stimulation of chondrocytes cultures with examples of bioreactorsthat can be used. [18]10
  13. 13. Measurement of cell responseAnother technique in cartilage tissue engineering is the measurements of cell deformation, whichhas been used to indirectly determine the material properties of chondrocytes. These measurementsare mostly performed within compressed gels in three ways: micropipette aspiration, cytoindentationand atomic force microscopy (Figure 2.7).Figure 2.7: Illustration of the three direct techniques currently used to characterize the biomechanicalcharacteristics: (A) micropipette aspiration, (B) cytoindentation and (C) atomic forcemicroscopy. [19]The majority of data on the properties of chondrocytes has been obtained using the micropipetteaspiration technique. A long range of measurements of the Young’s modulus and Poissons ratio ofchondrocytes has been performed, as well as volumetric properties and viscosity. These values vary dueto the experimental differences in the cells and scaffolds chosen, the stresses applied, and the methodof measurement. Measuring the rate at which chondrocytes can sense and respond to mechanicalstimuli can give valuable insight into intracellular regulatory mechanisms.Finally, the effects of various mechanical forces on gene expression are investigated. For example,static and intermittent hydrostatic pressure increased the expression of transforming growth factorβ, as well as aggrecan and type II collagen mRNA, in high-density monolayer cultures. In isolatedhuman chondrocytes grown in monolayer, constant fluid shear forces stimulated expression of mRNAfor tissue inhibitor of metalloproteinase. Dynamic mechanical forces have also been shown to influencematrix gene expression.2.3.3 Example of cell engineering mechanical stimulation studyTissue engineering of cartilage can have unusual applications such as the study of this tissue grow inspace [5]. This interest is mainly applicable for long term space flights, for passengers who are exposedto microgravity as well as launch and landing events. Results from these studies have enriched theunderstanding of the effects of pseudo-weightlessness on prolonged immobilization, hydrotherapy, andintrauterine development. These studies were conducted on the Mir Space Station and the results werecompared to those obtained by the same system on Earth. Specifically, three-dimensional cell-polymerconstructs consisting of bovine articular chondrocytes and polyglycolic acid scaffolds were grown inrotating bioreactors, first for 3 months on Earth and then for an additional 4 months on eitherMir (10−4-10−6 g) or Earth (1 g) as shown in figure 2.8. Chondrocytes were seeded onto scaffoldconstructs (5 million cells per PGA disc) in spinner flasks stirred at 80 rpm in a 37 ◦C humidified10% CO2 incubator. After 6 days, cell–PGA constructs were transferred into rotating bioreactorsconfigured as the annular space between a 5.75-cm diameter polycarbonate outer cylinder and a 2-cm diameter hollow inner cylinder. The entire vessel was rotated as a solid body around its centralaxis while gas exchange was provided by pumping incubator air through the inner cylinder. Culture11
  14. 14. medium was replaced at a rate of 50% every 3-4 days, and the vessel rotation speed was graduallyincreased from 15 to 28 rpm over 3 months, to induce mixing by gravitational construct settling. After3 months, constructs were transferred into each of two flight-qualified rotating, perfused bioreactors(BTS) for an additional 4 months of cultivation on either the Mir Space Station or on Earth. Bothenvironments yielded cartilaginous constructs, each weighing between 0.3 and 0.4 g and consisting ofviable, differentiated cells that synthesized proteoglycan and type II collagen. Compared with theEarth group, Mir grown constructs were smaller, more spherical, and mechanically inferior, as shownin figure 2.9. [5]Figure 2.8: Experimental design of the bioreactor.[5]Figure 2.9: Transmission electron micrograph of constructs from (A) Mir and (B) Earth. [5]12
  15. 15. 3 Future Developments: Microfluidics andMicrotechnologyThe emergence of microfluidics has inspired new strategies to more easily induce cellular mechan-otransduction [11]. Cellular stimulation by locally varying fluid shear can serve to accurately altermembrane surface tension as well as produce direct compressive and strain forces onto cells. More-over, microtechnology has increased the possibilities for individual cell-level actuation and readout.Microsystems are perfectly suited for the analyses of individual cells, as well as more complex cellculture and tissue systems, since the integrated tools match the length scales of cells (550 mm).Since small liquid volumes be controlled precisely, the cellular environment can be controlled quiteaccurately. Microsystems technology also offers the integration of microsized, movable, fast-operatingcomponents, such as valves , pumps, and microsized cell culture chambers. Figure 3.1 shows thesesort of features:Figure 3.1: Applications of µfluidic devices for mechanotransduction [11]Shear forces: The laminar flow regime that is achievable with microdevices facilitates the applicationof well-defined flow rates and profiles within the microfluidic channels and hence, allows for the localstimulation of cells with precise shear forces.Tensile stress: A thin membrane separating air-filled epithelial from the liquid-filled endothelialcompartments could be stretch-activated by adjacent vacuum channels, leading to a cyclical mechanicalstrain on the indicated cell types.Cell compression: Microfluidic cytometers have been developed to identify potentially cancerouscells based on their compression responses in a high throughput manner. By combining a polymercantilever-based method with micro restriction flow through, the cells mechanical response to physicalcompression could directly be measured.Intracellular architecture: Micropost arrays have also been successfully employed in more sophisti-cated designs. Clamping a stretchable polymeric membrane possessing a micropillar fabricated surfaceinto a cell-culturing module facilitated the measurement of intra-cellular microscopic forces with dy-namic extracellular microscopy.13
  16. 16. 4 ConclusionsMechanical stimulation plays an important role in the culture of cells, especially those that arenaturally under mechanical stress in the human body. When applied to cultivating cells, these inputsstart a cascade of signals that influence the development of the tissue. The relationship between theextracellular matrix and cell development, particularly the mechanical feedback process between them,is important for tissue engineers to understand in order to induce adequate mechanical stimulationto out-of-body environments. The many experiments that have involved mechanical stimulation havebecome a framework for upcoming studies. This report describes some but not all of them, since manyfactors are involved.The long term goal of tissue engineering is to develop in vitro tissues and organs with properties andbehaviors closely resembling those developed naturally in living beings. The behavior and developmentof cells provide vital clues to reaching this goal. Developing tools that allow researchers to bettermanipulate cells and mimic in vivo environments is a crucial step in the growth of this field. Thoughcurrent bioreactors successfully apply mechanotransduction to induce cell proliferation and grow 3Dgrafts, more research needs to be done to optimize the transplantation process. In addition, thoughmany present-day bioreactors provide adequate physical, chemical, and biological environments, afurther optimization of specific stress, load, and biophysical stimulus combinations is needed to refinegrowth processes according to the needs of specific cell types.14
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