Robert L. Mauck

Influence of seeding density and dynamic deformational loading on the developing structure/function relationships of chondrocyte-seeded agarose hydrogels.

AbstractChondrocytes cultured in agarose hydrogels develop a functional extracellular matrix. Application of dynamic strain at physiologic levels to these constructs over time can increase their mechanical properties. In this study, the effect of seeding density (20 and 60×106 cells/ml) on tissue elaboration was investigated. Higher seeding densities increased tissue properties in free-swelling culture, with constructs seeded at 20 and 60×106 cells/ml reaching maximum values over the 63 day culture period of aggregate modulus HA: 43±15 kPa, Young’s modulus EY: 39±3 kPa, and glycosaminglycan content [GAG]: 0.96%±0.13% wet weight; and HA: 58±12 kPa, EY: 60±5 kPa, and [GAG]: 1.49% ± 0.26% wet weight, respectively. It was further observed that the application of daily dynamic deformational loading to constructs seeded at 20×106 cells/ml enhanced biochemical content (∼150%) and mechanical properties (∼threefold) compared to free-swelling controls by day 28. However, at a concentration of 60×106 cells/ml, no difference in mechanical properties was found in loaded samples versus their free-swelling controls. Multiple regression analysis showed that the mechanical properties of the tissue constructs depend more strongly on collagen content than GAG content; a finding that is more pronounced with the application of daily dynamic deformational loading. Our findings provide evidence for initial cell seeding density and nutrient accessibility as important parameters in modulating tissue development of engineered constructs, and their ability to respond to a defined mechanical stimulus.

A Paradigm for Functional Tissue Engineering of Articular Cartilage via Applied Physiologic Deformational Loading

Deformational loading represents a primary component of the chondrocyte physical environment in vivo. This review summarizes our experience with physiologic deformational loading of chondrocyte-seeded agarose hydrogels to promote development of cartilage constructs having mechanical properties matching that of the parent calf tissue, which has a Youngs modulus EY = 277 kPa and unconfined dynamic modulus at 1 Hz G*=7 MPa. Over an 8-week culture period, cartilage-like properties have been achieved for 60 × 106 cells/ml seeding density agarose constructs, with EY = 186 kPa, G*=1.64 MPa. For these constructs, the GAG content reached 1.74% ww and collagen content 2.64% ww compared to 2.4% ww and 21.5% ww for the parent tissue, respectively. Issues regarding the deformational loading protocol, cell-seeding density, nutrient supply, growth factor addition, and construct mechanical characterization are discussed. In anticipation of cartilage repair studies, we also describe early efforts to engineer cylindrical and anatomically shaped bilayered constructs of agarose hydrogel and bone (i.e., osteochondral constructs). The presence of a bony substrate may facilitate integration upon implantation. These efforts will provide an underlying framework from which a functional tissue-engineering approach, as described by Butler and coworkers (2000), may be applied to general cell-scaffold systems adopted for cartilage tissue engineering.

Anatomically shaped osteochondral constructs for articular cartilage repair

Few successful treatment modalities exist for surface-wide, full-thickness lesions of articular cartilage. Functional tissue engineering offers a great potential for the clinical management of such lesions. Our long-term hypothesis is that anatomically shaped tissue constructs of entire articular layers can be engineered in vitro on a bony substrate, for subsequent implantation. To determine the feasibility, this study investigated the development of bilayered scaffolds of chondrocyte-seeded agarose on natural trabecular bone. In a series of three experiments, bovine chondrocytes were seeded in (1) cylindrical bilayered constructs of agarose and bovine trabecular bone, 0.53 cm2 in surface area and 3.2 mm thick, and were cultured for up to 6 weeks; (2) chondrocyte-seeded anatomically shaped agarose constructs reproducing the human patellar articular layer (area=11.7 cm2, mean thickness=3.4 mm), cultured for up to 6 weeks; and (3) chondrocyte-seeded anatomically shaped agarose constructs of the patella (same as above) integrated into a corresponding anatomically shaped trabecular bone substrate, cultured for up to 2 weeks. Articular layer geometry, previously acquired from human cadaver joints, was used in conjunction with computer-aided design and manufacturing technology to create these anatomically accurate molds. In all experiments, chondrocytes remained viable over the entire culture period, with the agarose maintaining its shape while remaining firmly attached to the underlying bony substrate (when present). With culture time, the constructs exhibited positive type II collagen staining as well as increased matrix elaboration (Safranin O staining for glycosaminoglycans) and material properties (Youngs modulus and aggregate modulus). Despite the use of relatively large agarose constructs partially integrated with trabecular bone, no adverse diffusion limitation effects were observed. Anatomically shaped constructs on a bony substrate may represent a new paradigm in the design of a functional articular cartilage tissue replacement.

Mitogen-activated protein kinase signaling in bovine articular chondrocytes in response to fluid flow does not require calcium mobilization

In the present study, the role of mitogen-activated protein kinases (MAPKs) in chondrocyte mechanotransduction was investigated. We hypothesized that MAPKs participate in fluid flow-induced chondrocyte mechanotransduction. To test our hypothesis, we studied cultured chondrocytes subjected to a well-defined mechanical stimulus generated with a laminar flow chamber. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) were activated 1.6-3-fold after 5-15 min of fluid flow exposure corresponding to a chamber wall shear stress of 1.6 Pa. Activation of ERK1/2 was observed in the presence of both 10% FBS and 0.1% BSA, suggesting that the flow effects do not require serum agonists. Treatment with thapsigargin or EGTA had no significant effect on the ERK1/2 activation response to flow, suggesting that Ca2+ mobilization is not required for this response. To assess downstream effects of the activated MAPKs on transcription, flow studies were performed using chondrocytes transfected with a chimeric luciferase construct containing 2.4 kb of the promoter region along with exon 1 of the human aggrecan gene. Two-hour exposure of transfected chondrocytes to fluid flow significantly decreased aggrecan promoter activity by 40%. This response was blocked by treatment of chondrocytes with the MEK-1 inhibitor PD98059. These findings demonstrate that, under the conditions of the present study, fluid flow-induced signals activate the MEK-1/ERK signaling pathway in articular chondrocytes, leading to down-regulation of expression of the aggrecan gene.

Osteocyte viability and regulation of osteoblast function in a 3D Trabecular bone explant under dynamic hydrostatic pressure

A new trabecular bone explant model was used to examine osteocyte‐osteoblast interactions under DHP loading. DHP loading enhanced osteocyte viability as well as osteoblast function measured by osteoid formation. However, live osteocytes were necessary for osteoblasts to form osteoids in response to DHP, which directly show osteoblast‐osteocyte interactions in this in vitro culture.

The role of osmotic pressure and tension-compression nonlinearity in the frictional response of articular cartilage

Articular cartilage is the bearing material of diarthrodial joints such as the knee, hip, or shoulder. Some studies of cartilage lubrication have hypothesized that pressurization of its interstitial fluid may contribute predominantly to reducing the friction coefficient at the contact interface of articular layers. This study introduces a formulation for the dependence of the frictional response of articular cartilage on interstitial fluid pressurization, which accounts for the osmotic pressure in cartilage as well as the tissues tension-compression nonlinearity, and is based on the theory of mixtures for soft hydrated charged tissues. Theoretical predictions of this model are obtained for the configuration of unconfined compression creep. It is observed from theory that increasing the salt concentration of the tissues bathing solution reduces the minimum friction coefficient that can be achieved, relative to its equilibrium value; the model also predicts that increasing the applied load can similarly reduce the minimum friction coefficient. Physical interpretations of these phenomena are provided by the model. Experimental results are presented which support these theoretical findings and produce time-dependent responses in good agreement with model predictions. Furthermore, it is observed that the equilibrium friction coefficient does not remain constant under various loads or salt concentrations, and correlation analyses suggest that the equilibrium value depends in part on the compressive strain in the tissue.

Cartilage Interstitial Fluid Load Support in Unconfined Compression Following Enzymatic Digestion

Interstitial fluid pressurization plays an important role in cartilage biomechanics and is believed to be a primary mechanism of load support in synovial joints. The objective of this study was to investigate the effects of enzymatic degradation on the interstitial fluid load support mechanism of articular cartilage in unconfined compression. Thirty-seven immature bovine cartilage plugs were tested in unconfined compression before and after enzymatic digestion. The peak fluid load support decreased significantly (p < 0.0001) from 84 +/- 10% to 53 +/- 19% and from 80 +/- 10% to 46 +/- 21% after 18-hours digestion with 1.0 u/mg-wet-weight and 0.7 u/mg-wet-weight of collagenase, respectively. Treatment with 0.1 u/ml of chondroitinase ABC for 24 hours also significantly reduced the peak fluid load support from 83 +/- 12% to 48 +/- 16% (p < 0.0001). The drop in interstitial fluid load support following enzymatic treatment is believed to result from a decrease in the ratio of tensile to compressive moduli of the solid matrix.

Intermittent Hydrostatic Pressurization Modulates Gene Expression in Human Dermal Fibroblasts Seeded in Three-Dimensional Polymer Scaffolds

Mechanical stimuli are known to regulate the morphology and differentiated function of connective tissue cells. In particular, hydrostatic pressure has been reported to alter cytoskeletal organization in osteoblast-like cells (1) and chondrocytes (2), and to modulate metabolic activity in both chondrocytes (3–5) and intervertebral disc cells (6). The cellular response to continuous hydrostatic pressure is generally catabolic (3) while intermittent hydrostatic pressure at frequencies ranging from 0.25–1.0 Hz (3–5) is anabolic, giving rise to increased expression and biosynthesis of extracellular matrix (ECM) components. Previously, human dermal fibroblasts in monolayer culture were shown to respond to hydrostatic pressure by increasing heat shock protein expression levels (7). In this study, we characterize the effects of intermittent hydrostatic pressure on gene expression in human dermal fibroblasts seeded in three-dimensional polymer scaffolds.Copyright