Cellular and Molecular Bioengineering: A Tipping Point.

In January of 2011, the Biomedical Engineering Society (BMES) and the Society for Physical Regulation in Biology and Medicine (SPRBM) held its inaugural Cellular and Molecular Bioengineering (CMBE) conference. The CMBE conference assembled worldwide leaders in the field of CMBE and held a very successful Round Table discussion among leaders. One of the action items was to collectively construct a white paper regarding the future of CMBE. Thus, the goal of this report is to emphasize the impact of CMBE as an emerging field, identify critical gaps in research that may be answered by the expertise of CMBE, and provide perspectives on enabling CMBE to address challenges in improving human health. Our goal is to provide constructive guidelines in shaping the future of CMBE.

A hydrogel-mineral composite scaffold for osteochondral interface tissue engineering.

Osteoarthritis is the leading cause of physical disability among Americans, and tissue engineered cartilage grafts have emerged as a promising treatment option for this debilitating condition. Currently, the formation of a stable interface between the cartilage graft and subchondral bone remains a significant challenge. This study evaluates the potential of a hybrid scaffold of hydroxyapatite (HA) and alginate hydrogel for the regeneration of the osteochondral interface. Specifically, the effects of HA on the response of chondrocytes were determined, focusing on changes in matrix production and mineralization, as well as scaffold mechanical properties over time. Additionally, the optimal chondrocyte population for interface tissue engineering was evaluated. It was observed that the HA phase of the composite scaffold promoted the formation of a proteoglycan- and type II collagen-rich matrix when seeded with deep zone chondrocytes. More importantly, the elevated biosynthesis translated into significant increases in both compressive and shear moduli relative to the mineral-free control. Presence of HA also promoted chondrocyte hypertrophy and type X collagen deposition. These results demonstrate that the hydrogel-calcium phosphate composite supported the formation of a calcified cartilage-like matrix and is a promising scaffold design for osteochondral interface tissue engineering.

Matrix deposition modulates the viscoelastic shear properties of hydrogel-based cartilage grafts.

Hydrogel-based scaffolds such as alginate have been extensively investigated for cartilage tissue engineering, largely due to their biocompatibility, ambient gelling conditions, and the ability to support chondrocyte phenotype. While it is well established that the viscoelastic response of articular cartilage is essential for articulation and load bearing, the time-dependent mechanical properties of hydrogel-based cartilage scaffolds have not been extensively studied. Therefore, the objective of this study was to characterize the intrinsic viscoelastic shear properties of chondrocyte-laden alginate scaffolds and determine the effects of seeding density and culturing time on these properties. Specifically, the viscoelastic properties (equilibrium and dynamic shear moduli and dynamic phase shift angle) of these engineered cartilage grafts were measured under torsional shear. In addition, the rapid ramp-step shear stress relaxation of the alginate-based cartilage scaffolds was modeled using the quasi-linear viscoelastic (QLV) theory. It was found that scaffold stiffness increased with both culturing time and cell density, whereas viscosity did not change significantly with cell density (30 vs. 60 million/mL). Similar to native cartilage, the energy dissipation of engineered scaffolds under pure shear is highly correlated to the glycosaminoglycan content. In contrast, collagen content was not strongly correlated to scaffold shear modulus, especially the instantaneous shear modulus predicted by the quasi-linear viscoelastic model. The findings of this study provide new insights into the structure-function relationship of engineered cartilage and design of functional grafts for cartilage repair.

Geometric control of human stem cell morphology and differentiation.

During tissue morphogenesis, stem cells and progenitor cells migrate, proliferate, and differentiate, with striking changes in cell shape, size, and acting mechanical stresses. The local cellular function depends on the spatial distribution of cytokines as well as local mechanical microenvironments in which the cells reside. In this study, we controlled the organization of human adipose derived stem cells using micro-patterning technologies, to investigate the influence of multi-cellular form on spatial distribution of cellular function at an early stage of cell differentiation. The underlying role of cytoskeletal tension was probed through drug treatment. Our results show that the cultivation of stem cells on geometric patterns resulted in pattern- and position-specific cell morphology, proliferation and differentiation. The highest cell proliferation occurred in the regions with large, spreading cells (such as the outer edge of a ring and the short edges of rectangles). In contrast, stem cell differentiation co-localized with the regions containing small, elongated cells (such as the inner edge of a ring and the regions next to the short edges of rectangles). The application of drugs that inhibit the formation of actomyosin resulted in the lack of geometrically specific differentiation patterns. This study confirms the role of substrate geometry on stem cell differentiation, through associated physical forces, and provides a simple and controllable system for studying biophysical regulation of cell function.

A triphasic orthotropic laminate model for cartilage curling behavior: fixed charge density versus mechanical properties inhomogeneity.

Osmotic pressure and associated residual stresses play important roles in cartilage development and biomechanical function. The curling behavior of articular cartilage was believed to be the combination of results from the osmotic pressure derived from fixed negative charges on proteoglycans and the structural and compositional and material property inhomogeneities within the tissue. In the present study, the in vitro swelling and curling behaviors of thin strips of cartilage were analyzed with a new structural model using the triphasic mixture theory with a collagen-proteoglycan solid matrix composed of a three-layered laminate with each layer possessing a distinct set of orthotropic properties. A conewise linear elastic matrix was also incorporated to account for the well-known tension-compression nonlinearity of the tissue. This model can account, for the first time, for the swelling-induced curvatures found in published experimental results on excised cartilage samples. The results suggest that for a charged-hydrated soft tissue, such as articular cartilage, the balance of proteoglycan swelling and the collagen restraining within the solid matrix is the origin of the in situ residual stress, and that the layered collagen ultrastructure, e.g., relatively dense and with high stiffness at the articular surface, play the dominate role in determining curling behaviors of such tissues.

A linearized formulation of triphasic mixture theory for articular cartilage, and its application to indentation analysis.

The negative charges on proteoglycans significantly affect the mechanical behaviors of articular cartilage. Mixture theories, such as the triphasic theory, can describe quantitatively how this charged nature contributes to the mechano-electrochemical behaviors of such tissue. However, the mathematical complexity of the theory has hindered its application to complicated loading profiles, e.g., indentation or other multi-dimensional configurations. In this study, the governing equations of triphasic mixture theory for soft tissue were linearized and dramatically simplified by using a regular perturbation method and the use of two potential functions. We showed that this new formulation can be used for any axisymmetric problem, such as confined or unconfined compressions, hydraulic perfusion, and indentation. A finite difference numerical program was further developed to calculate the deformational, electrical, and flow behaviors inside the articular cartilage under indentation. The calculated tissue response was highly consistent with the data from indentation experiments (our own and those reported in the literature). It was found that the charged nature of proteoglycans can increase the apparent stiffness of the solid matrix and lessen the viscous effect introduced by fluid flow. The effects of geometric and physical properties of indenter tip, cartilage thickness, and that of the electro-chemical properties of cartilage on the resulting deformation and fluid pressure fields across the tissue were also investigated and presented. These results have implications for studying chondrocyte mechanotransduction in different cartilage zones and for tissue engineering designs or in vivo cartilage repair.

The impact of biomechanics in tissue engineering and regenerative medicine.

Biomechanical factors profoundly influence the processes of tissue growth, development, maintenance, degeneration, and repair. Regenerative strategies to restore damaged or diseased tissues in vivo and create living tissue replacements in vitro have recently begun to harness advances in understanding of how cells and tissues sense and adapt to their mechanical environment. It is clear that biomechanical considerations will be fundamental to the successful development of clinical therapies based on principles of tissue engineering and regenerative medicine for a broad range of musculoskeletal, cardiovascular, craniofacial, skin, urinary, and neural tissues. Biomechanical stimuli may in fact hold the key to producing regenerated tissues with high strength and endurance. However, many challenges remain, particularly for tissues that function within complex and demanding mechanical environments in vivo. This paper reviews the present role and potential impact of experimental and computational biomechanics in engineering functional tissues using several illustrative examples of past successes and future grand challenges.

Temperature-dependent viscoelastic properties of the human supraspinatus tendon.

Temperature effects on the viscoelastic properties of the human supraspinatus tendon were investigated using static stress-relaxation experiments and the quasi-linear viscoelastic (QLV) theory. Twelve supraspinatus tendons were randomly assigned to one of two test groups for tensile testing using the following sequence of temperatures: (1) 37, 27, and 17 degrees C (Group I, n=6), or (2) 42, 32, and 22 degrees C (Group II, n=6). QLV parameter C was found to increase at elevated temperatures, suggesting greater viscous mechanical behavior at higher temperatures. Elastic parameters A and B showed no significant difference among the six temperatures studied, implying that the viscoelastic stress response of the supraspinatus tendon is not sensitive to temperature over shorter testing durations. Using regression analysis, an exponential relationship between parameter C and test temperature was implemented into QLV theory to model temperature-dependent viscoelastic behavior. This modified approach facilitates the theoretical determination of the viscoelastic behavior of tendons at arbitrary temperatures.

Biomechanics of articular cartilage and determination of material properties.

Descriptions of the mechanical behaviors of articular cartilage and their correlations with collagen, proteoglycan, water, and ions are summarized, with particular emphasis on understanding the osmotic effect inside the tissue. First, a descriptive explanation is presented of the biphasic theory required to understand how interstitial water contributes toward the viscoelastic behavior of any hydrated soft tissue. Then, the famous osmotic effect in charged, hydrated soft tissue is interpreted in light of the triphasic mixture theory framework. In the introduction of mechanical testing methods, our emphasis is on the popular indentation technique, which can determine the material properties of cartilage in situ or in vivo. The widely accepted indentation analysis solutions in cartilage biomechanics history are summarized and evaluated. At the end of this paper, a new generalized correspondence principle between charged, hydrated soft tissue and linear, isotropic, elastic material (i.e., elasticity theory) is introduced. This principle makes the employment of triphasic theory as straightforward as using an elasticity theory to solve any equilibrium problem where the elasticity theory can be used to model the material. By using this generalized correspondence principle, the fixed charge density of bovine cartilage has been simply and conveniently calculated from the indentation testing data. The results of proteoglycan content from this mechanical test are remarkably consistent with those from standard biochemical assay. This new correspondence principle significantly improves the power of indentation tests in the determination of mechanoelectrochemical properties of articular cartilage.

Calcium Concentration Effects on the Mechanical and Biochemical Properties of Chondrocyte-Alginate Constructs.

Alginate gel crosslinked by calcium ions (Ca(2+)) has been widely used in cartilage tissue engineering. However, most studies have been largely performed in vitro in medium with a calcium concentration ([Ca(2+)]) of 1.8mM, while the calcium level in the synovial fluid of the human knee joints, for example, has been reported to be 4mM or even higher. To simulate the synovial environment, the two studies in this paper were designed to investigate how the alginate scaffold alone, as well as the chondrocytes seeded alginate gel responds to variations in medium [Ca(2+)]. In Study A, the mechanical properties of 2% alginate hydrogel were tested in 0.15M NaCl and various [Ca(2+)] (1.0mM, 1.8mM, and 4mM). In Study B, primary bovine chondrocytes was seeded in alginate gel, and biochemical contents and mechanical properties were determined after incubation for 28 days in three [Ca(2+)] (1.8mM, 4mM, and 8mM). For both studies, it was found that the magnitude of the complex shear modulus (|G*|) at 1Hz doubled and the corresponding phase angle shift angle (δ) increased > 2° as a result of the approximate 4-fold change in [Ca(2+)]. At high [Ca(2+)], the chondrocyte glycosaminogylcan (GAG) production inside the chondrocyte-alginate constructs was suppressed significantly. This is likely due to a decrease in the porosity of the chondrocyte-alginate constructs as a result of compaction in structure caused by an increased crosslinking density with [Ca(2+)]. These may be important considerations in the eventual successful implementation of cartilage tissue-engineered constructs in the clinical setting.

The generalized triphasic correspondence principle for simultaneous determination of the mechanical properties and proteoglycan content of articular cartilage by indentation.

The triphasic mixture theory has been used to describe the mechanical and physicochemical behaviors of articular cartilage under some specialized loading conditions. However, the mathematical complexities of this theory have limited its applications for theoretical analyses of experimental studies and models for predicting cartilage and other biological tissues' deformational behaviors. A generalized correspondence principle has been established in the present study, and this principle shows that the equilibrium deformational behavior of a charged-hydrated material under loading is identical to that of an elastic medium without charge. A set of explicit formulas has been derived to correlate the mechanical properties of an equivalent material with the intrinsic elastic moduli, fixed charge density and free-ion concentration within the cartilage tissue. The validity of these formulas is independent of the deformation state of the elastic solid matrix under an infinitesimal strain. Therefore they can be employed for any loading conditions, such as confined or unconfined compression, tension, and indentation tests, etc. In the current study, the fixed charge density of bovine cartilage is determined from the indentation creep data using this generalized correspondence principle. The proteoglycan content results were then compared with those from biochemical assay, yielding a linear regression slope of 1.034. Additionally a correspondence principle within a framework of cubic symmetry and a bilinear response in tension-compression (the conewise linear elasticity model) has also been developed to demonstrate the potential application of current methodology for inhomogeneous, anisotropic and nonlinear situations.

The inferior glenohumeral ligament: a correlative investigation.

The inferior glenohumeral ligament (IGHL) was investigated by correlating the biomechanical properties, biochemical composition, and histologic morphology of its 3 anatomic regions (superior band, anterior axillary pouch, and posterior axillary pouch) in 8 human cadaveric shoulders. The overall biochemical composition of the IGHL appeared similar to other ligaments, with average water content of 80.9 +/- 2.5%, collagen content of 80.0 +/- 9.2%, and crosslinks of 0.715 +/- 0.13 mol/mol collagen. The proteoglycan content was highest in the superior band (2.73 +/- 0.7 mg/g dry weight) and may, in part, explain its viscoelastic behavior. Histologic analysis demonstrated longitudinally organized fiber bundles that were more uniform in the mid-substance but more interwoven and less uniformly oriented near the insertion sites. The superior band had the most pronounced fiber bundle interweaving, while crimping was more evident in the anterior axillary pouch. Elastin was identified in each of the regions. Tensile testing demonstrated a trend toward higher ultimate tensile stress (16.9 +/- 7.9 MPa) and tensile modulus (130.3 +/- 47.9 MPa) in the superior band compared to the axillary pouch. The mean ultimate tensile strain of the IGHL was 16.8 +/- 4.6%. These complex IGHL properties may help to explain its unique functions in stabilizing the shoulder in different arm positions and at different rates of loading, including the failure patterns seen clinically, as in Bankart lesions (insertion site) versus capsular stretching (ligament substance).

An in vitro analysis of ligament reconstruction or extension osteotomy on trapeziometacarpal joint stability and contact area.

PURPOSE: Painful instability of the minimally osteoarthritic thumb carpometacarpal (CMC) joint can be treated successfully by either ligament reconstruction or metacarpal extension osteotomy. The purpose of this study was to measure the laxity of cadaveric thumb CMC joints and to determine the influence of ligament reconstruction and metacarpal osteotomy on joint laxity and contact area. METHODS: The baseline laxity of CMC joints from 25 fresh-frozen human cadaveric specimens (average age, 42 y; range, 18-55 y) was measured in the position of lateral pinch on a custom-designed CMC joint laxity tester. Joint laxity was measured again after 2 surgical simulations consisting of either a metacarpal extension osteotomy (at 10 degrees and 15 degrees) or a simulated Eaton-Littler ligament reconstruction (including total, volar, and dorsal ligament reconstructions relative to the plane of the thumbnail). Contact area between the thumb metacarpal and trapezium during testing was determined using stereophotogrammetry. RESULTS: The 15 degrees extension osteotomy significantly reduced CMC joint laxity in the radial-ulnar, dorsal-volar, pronation-supination, and distraction directions in the position of lateral pinch. The 10 degrees osteotomy reduced laxity only in the dorsal-volar direction. The total ligament reconstruction significantly reduced joint laxity in the radial-ulnar, dorsal-volar, and pronation-supination directions. The dorsal ligament reconstruction reduced laxity in the dorsal-volar direction only; the volar ligament reconstruction reduced laxity in both dorsovolar and radioulnar directions. The 10 degrees and 15 degrees osteotomies produced a dorsal shift of the weighted centroid of contact on the metacarpal and trapezium, whereas the ligament reconstruction did not produce such an effect. CONCLUSIONS: In the position of lateral pinch the 15 degrees osteotomy and total ligament reconstruction significantly reduced laxity of the thumb CMC joint in all directions tested. The isolated dorsal or volar ligament reconstructions both reduced dorsal-volar laxity. Metacarpal extension osteotomy may stabilize the thumb CMC joint in lateral pinch to a degree similar to that of a standard ligament reconstruction.

Tissue engineering and developmental biology: going biomimetic.

This article contains the collective views expressed at the first session of the workshop "Tissue Engineering--The Next Generation," which was devoted to the interactions between developmental biology and tissue engineering. Donald Ingber discussed the chasms between developmental biology and tissue engineering from the perspective of a cell biologist who has had interest in tissue engineering since its early days. Van C. Mow shared a historical perspective on the development of tissue engineering as one of the first engineers involved in the field. David Butler offered an assessment of functional tissue engineering, a new area he helped establish and promote. Laura Niklason discussed how to be more effective in developing cellular therapies for large numbers of patients. Johnny Huard described his approach to tissue engineering, based on the use of muscle-derived cells. Jeremy Mao focused on cell homing and cell density in the context of native development and relevance to tissue engineering. Ioannis Yannas proposed a set of "rules" in organ regeneration. Collectively, the faculty expressed a remarkable level of enthusiasm for bridging the gaps between developmental biology and tissue engineering and offered new ideas on how to facilitate the interaction between the two fields.

Inhomogeneous mechanical behavior of the human supraspinatus tendon under uniaxial loading.

Disorders of the rotator cuff, particularly tears of the rotator cuff tendons, cause significant shoulder disability. Among numerous factors thought to be responsible for the initiation and progression of supraspinatus tears are those related to the tendon's biomechanical properties. We hypothesized that in supraspinatus tendons subjected to tensile loading a strain gradient (difference) exists between the articular and bursal tendon surfaces, that regional strain differences exist on each of these two tendon surfaces, and that tendon surface strains vary with glenohumeral abduction. To test these hypotheses, the intrinsic inhomogeneous deformational characteristics of the articular and bursal surfaces of eight intact human cadaveric supraspinatus tendons were studied at three glenohumeral abduction angles using a novel multiple strain measuring system which simultaneously recorded surface marker displacements on two opposing soft tissue surfaces. Under applied tensile loads, the articular surface exhibited greater strain at 22 degrees (7.4+/-2.6% vs. 1.3+/-0.7%, p=0.0002) and 63 degrees (6.4+/-1.6% vs. 2.7+/-1.2%, p=0.0001) whereas the bursal surface exhibited greater strain at 90 degrees (7.6+/-2.8% vs. 4.9+/-0.4%, p=0.013). At all abduction angles, insertion strains were higher than those of the mid-tendon and tendon-muscle junction regions. The existence of inhomogeneous surface strains in the intact supraspinatus tendon demonstrates that intratendinous shear occurs within the tendon. The higher strain on the articular side of the tendon, especially at the insertion region, suggests a propensity for tears to initiate in the articular tendinous zone.

Comparison of glenohumeral mechanics following a capsular shift and anterior tightening.

BACKGROUND: Numerous surgical techniques have been developed to treat glenohumeral instability. Anterior tightening procedures have been associated with secondary glenohumeral osteoarthritis, unlike the anterior-inferior capsular shift procedure, which has been widely advocated as a more anatomical repair. The objective of the present study was to quantify glenohumeral joint translations, articular contact, and resultant forces in cadaveric specimens in order to compare the effects of unidirectional anterior tightening with those of the anterior-inferior capsular shift. METHODS: Six normal fresh-frozen cadaveric shoulders were tested on a custom rig with use of a coordinate-measuring machine to obtain kinematic measurements and a six-axis load transducer to measure resultant external joint forces. Shoulders were tested in the scapular plane in three configurations (normal anatomical, anterior tightening, and anterior-inferior capsular shift) and in three humeral rotations (neutral, internal, and external). Glenohumeral articular surface geometry was quantified with use of stereophotogrammetry for kinematic and contact analyses. Resultant joint forces were computed on the basis of digitized coordinates of tendon insertions and origins. RESULTS: Compared with the controls (maximum elevation, 167 degrees 8 degrees ), the anteriorly tightened specimens demonstrated loss of external rotation, significantly restricted maximum elevation (135 degrees 16 degrees , p = 0.002), posterior-inferior humeral head subluxation, and significantly greater posteriorly directed resultant forces at higher elevations (p < 0.05). In contrast, compared with the controls, the specimens that had been treated with the anterior-inferior capsular shift demonstrated a similar maximum elevation (159 degrees +/- 11 degrees , p = 0.8) without any apparent loss of external rotation and with reduced humeral translation. CONCLUSIONS: Anterior tightening adversely affects joint mechanics by decreasing joint stability, limiting both external rotation and arm elevation, and requiring greater posterior joint forces to attain maximum elevation. The anterior-inferior capsular shift improves joint stability while preserving external rotation with no significant loss of maximum elevation.

Anisotropy, inhomogeneity, and tension-compression nonlinearity of human glenohumeral cartilage in finite deformation.

The tensile and compressive properties of human glenohumeral cartilage were determined by testing 120 rectangular strips in uniaxial tension and 70 cylindrical plugs in confined compression, obtained from five human glenohumeral joints. Specimens were harvested from five regions across the articular surface of the humeral head and two regions on the glenoid. Tensile strips were obtained along two orientations, parallel and perpendicular to the split-line directions. Two serial slices through the thickness, corresponding to the superficial and middle zones of the cartilage layers, were prepared from each tensile strip and each compressive plug. The equilibrium tensile modulus and compressive aggregate modulus of cartilage were determined from the uniaxial tensile and confined compression tests, respectively. Significant differences in the tensile moduli were found with depth and orientation relative to the local split-line direction. Articular cartilage of the humeral head was significantly stiffer in tension than that of the glenoid. There were significant differences in the aggregate compressive moduli of articular cartilage between superficial and middle zones in the humeral head. Furthermore, tensile and compressive stress-strain responses exhibited nonlinearity under finite strain, while the tensile modulus differed by up to two orders of magnitude from the compressive aggregate modulus at 0% strain, demonstrating a high degree of tension-compression nonlinearity. The complexity of the mechanical properties of human glenohumeral cartilage was exposed in this study, showing anisotropy, inhomogeneity, and tension-compression nonlinearity within the same joint. The observed differences in the tensile properties of human glenohumeral cartilage suggest that the glenoid may be more susceptible to cartilage degeneration than the humeral head.