Bendable osteochondral allografts for patellar resurfacing: A finite element analysis of congruence.

Osteochondral allograft (OCA) transplantation provides a safe and effective treatment option for large cartilage defects, but its use is limited partly due to the difficulty of matching articular surface curvature between donor and recipient. We hypothesize that bendable OCAs may provide better curvature matching for patella transplants in the patellofemoral joint (PFJ). This finite element study investigates PFJ congruence for unbent and bendable OCAs, at various flexion angles. Finite element models were created for 12 femur-patella OCA pairings. Two grooves were cut in each OCA bony substrate, allowing the articular layer to bend. PFJs with either unbent (OCA) or permanently bent (BOCA) allografts were articulated from 40 to 70 degrees flexion and contact area was calculated. OCAs and BOCAs were then shifted 6 mm distally toward the tibia (S-OCA, S-BOCA) to investigate the influence of proximal-distal alignment on congruence. On average, no significant difference in contact area was found between native PFJs and either OCAs or BOCAs (p > 0.25), indicating that both types of allografts restored native congruence. This result provides biomechanical support in favor of an emerging surgical procedure. S-BOCAs resulted in a significant increase in contact area relative to the remaining groups (p < 0.02). The fact that BOCAs produced equally good results implies that bendable allografts may prove useful in future surgical procedures, with the possibility of transplanting them with a small distal shift. Surgeons who are reluctant to use OCAs for resurfacing patellae based on curvature matching capabilities may be more amenable to adopting BOCAs.

Sustained low-dose dexamethasone delivery via a PLGA microsphere-embedded agarose implant for enhanced osteochondral repair.

Articular cartilage defects are a common source of joint pain and dysfunction. We hypothesized that sustained low-dose dexamethasone (DEX) delivery via an acellular osteochondral implant would have a dual pro-anabolic and anti-catabolic effect, both supporting the functional integrity of adjacent graft and host tissue while also attenuating inflammation caused by iatrogenic injury. An acellular agarose hydrogel carrier with embedded DEX-loaded poly(lactic-co-glycolic) acid (PLGA) microspheres (DLMS) was developed to provide sustained release for at least 99 days. The DLMS implant was first evaluated in an in vitro pro-inflammatory model of cartilage degradation. The implant was chondroprotective, as indicated by maintenance of Young's modulus (EY) (p = 0.92) and GAG content (p = 1.0) in the presence of interleukin-1ß insult. In a subsequent preliminary in vivo experiment, an osteochondral autograft transfer was performed using a pre-clinical canine model. DLMS implants were press-fit into the autograft donor site and compared to intra-articular DEX injection (INJ) or no DEX (CTL). Functional scores for DLMS animals returned to baseline (p = 0.39), whereas CTL and INJ remained significantly worse at 6 months (p < 0.05). DLMS knees were significantly more likely to have improved OARSI scores for proteoglycan, chondrocyte, and collagen pathology (p < 0.05). However, no significant improvements in synovial fluid cytokine content were observed. In conclusion, utilizing a targeted DLMS implant, we observed in vitro chondroprotection in the presence of IL-1-induced degradation and improved in vivo functional outcomes. These improved outcomes were correlated with superior histological scores but not necessarily a dampened inflammatory response, suggesting a primarily pro-anabolic effect. STATEMENT OF SIGNIFICANCE: Articular cartilage defects are a common source of joint pain and dysfunction. Effective treatment of these injuries may prevent the progression of osteoarthritis and reduce the need for total joint replacement. Dexamethasone, a potent glucocorticoid with concomitant anti-catabolic and pro-anabolic effects on cartilage, may serve as an adjuvant for a variety of repair strategies. Utilizing a dexamethasone-loaded osteochondral implant with controlled release characteristics, we demonstrated in vitro chondroprotection in the presence of IL-1-induced degradation and improved in vivo functional outcomes following osteochondral repair. These improved outcomes were correlated with superior histological cartilage scores and minimal-to-no comorbidity, which is a risk with high dose dexamethasone injections. Using this model of cartilage restoration, we have for the first time shown the application of targeted, low-dose dexamethasone for improved healing in a preclinical model of focal defect repair.

Dynamic loading of deformable porous media can induce active solute transport.

Active solute transport mediated by molecular motors across porous membranes is a well-recognized mechanism for transport across the cell membrane. In contrast, active transport mediated by mechanical loading of porous media is a non-intuitive mechanism that has only been predicted recently from theory, but not yet observed experimentally. This study uses agarose hydrogel and dextran molecules as a model experimental system to explore this mechanism. Results show that dynamic loading can enhance the uptake of dextran by a factor greater than 15 over passive diffusion, for certain combinations of gel concentration and dextran molecular weight. Upon cessation of loading, the concentration reverts back to that achieved under passive diffusion. Thus, active solute transport in porous media can indeed be mediated by cyclical mechanical loading.

* Constrained Cage Culture Improves Engineered Cartilage Functional Properties by Enhancing Collagen Network Stability.

When cultured with sufficient nutrient supply, engineered cartilage synthesizes proteoglycans rapidly, producing an osmotic swelling pressure that destabilizes immature collagen and prevents the development of a robust collagen framework, a hallmark of native cartilage. We hypothesized that mechanically constraining the proteoglycan-induced tissue swelling would enhance construct functional properties through the development of a more stable collagen framework. To test this hypothesis, we developed a novel "cage" growth system to mechanically prevent tissue constructs from swelling while ensuring adequate nutrient supply to the growing construct. The effectiveness of constrained culture was examined by testing constructs embedded within two different scaffolds: agarose and cartilage-derived matrix hydrogel (CDMH). Constructs were seeded with immature bovine chondrocytes and cultured under free swelling (FS) conditions for 14 days with transforming growth factor-ß before being placed into a constraining cage for the remainder of culture. Controls were cultured under FS conditions throughout. Agarose constructs cultured in cages did not expand after the day 14 caging while FS constructs expanded to 8 × their day 0 weight after 112 days of culture. In addition to the physical differences in growth, by day 56, caged constructs had higher equilibrium (agarose: 639 ± 179 kPa and CDMH: 608 ± 257 kPa) and dynamic compressive moduli (agarose: 3.4 ± 1.0 MPa and CDMH 2.8 ± 1.0 MPa) than FS constructs (agarose: 193 ± 74 kPa and 1.1 ± 0.5 MPa and CDMH: 317 ± 93 kPa and 1.8 ± 1.0 MPa for equilibrium and dynamic properties, respectively). Interestingly, when normalized to final day wet weight, cage and FS constructs did not exhibit differences in proteoglycan or collagen content. However, caged culture enhanced collagen maturation through the increased formation of pyridinoline crosslinks and improved collagen matrix stability as measured by α-chymotrypsin solubility. These findings demonstrate that physically constrained culture of engineered cartilage constructs improves functional properties through improved collagen network maturity and stability. We anticipate that constrained culture may benefit other reported engineered cartilage systems that exhibit a mismatch in proteoglycan and collagen synthesis.

Dexamethasone Release from Within Engineered Cartilage as a Chondroprotective Strategy Against Interleukin-1α.

While significant progress has been made toward engineering functional cartilage constructs with mechanical properties suitable for in vivo loading, the impact on these grafts of inflammatory cytokines, chemical factors that are elevated with trauma or osteoarthritis, is poorly understood. Previous work has shown dexamethasone to be a critical compound for cultivating cartilage with functional properties, while also providing chondroprotection from proinflammatory cytokines. This study tested the hypothesis that the incorporation of poly(lactic-co-glycolic acid) (PLGA) (75:25) microspheres that release dexamethasone from within chondrocyte-seeded agarose hydrogel constructs would promote development of constructs with functional properties and protect constructs from the deleterious effects of interleukin-1α (IL-1α). After 28 days of growth culture, experimental groups were treated with IL-1α (10 ng/mL) for 7 days. Reaching native equilibrium moduli and proteoglycan levels, dexamethasone-loaded microsphere constructs exhibited tissue properties similar to microsphere-free control constructs cultured in dexamethasone-supplemented culture media and were insensitive to IL-1α exposure. These findings are in stark contrast to constructs containing dexamethasone-free microspheres or no microspheres, cultured without dexamethasone, where IL-1α exposure led to significant tissue degradation. These results support the use of dexamethasone delivery from within engineered cartilage, through biodegradable microspheres, as a strategy to produce mechanically functional tissues that can also combat the deleterious effects of local proinflammatory cytokine exposure.

Biocompatibility of polysebacic anhydride microparticles with chondrocytes in engineered cartilage.

One of main challenges in developing clinically relevant engineered cartilage is overcoming limited nutrient diffusion due to progressive elaboration of extracellular matrix at the periphery of the construct. Macro-channels have been used to decrease the nutrient path-length; however, the channels become occluded with matrix within weeks in culture, reducing nutrient diffusion. Alternatively, microparticles can be imbedded throughout the scaffold to provide localized nutrient delivery. In this study, we evaluated biocompatibility of polysebacic anhydride (PSA) polymers and the effectiveness of PSA-based microparticles for short-term delivery of nutrients in engineered cartilage. PSA-based microparticles were biocompatible with juvenile bovine chondrocytes for concentrations up to 2mg/mL; however, cytotoxicity was observed at 20mg/mL. Cytotoxicity at high concentrations is likely due to intracellular accumulation of PSA degradation products and resulting lipotoxicity. Cytotoxicity of PSA was partially reversed in the presence of bovine serum albumin. In conclusion, the findings from this study demonstrate concentration-dependent biocompatibility of PSA-based microparticles and potential application as a nutrient delivery vehicle that can be imbedded in scaffolds for tissue engineering.

Wear and damage of articular cartilage with friction against orthopedic implant materials.

The objective of this study was to measure the wear response of immature bovine articular cartilage tested against glass or alloys used in hemiarthroplasties. Two cobalt chromium alloys and a stainless steel alloy were selected for these investigations. The surface roughness of one of the cobalt chromium alloys was also varied within the range considered acceptable by regulatory agencies. Cartilage disks were tested in a configuration that promoted loss of interstitial fluid pressurization to accelerate conditions believed to occur in hemiarthroplasties. Results showed that considerably more damage occurred in cartilage samples tested against stainless steel (10 nm roughness) and low carbon cobalt chromium alloy (27 nm roughness) compared to glass (10 nm) and smoother low or high carbon cobalt chromium (10 nm). The two materials producing the greatest damage also exhibited higher equilibrium friction coefficients. Cartilage damage occurred primarily in the form of delamination at the interface between the superficial tangential zone and the transitional middle zone, with much less evidence of abrasive wear at the articular surface. These results suggest that cartilage damage from frictional loading occurs as a result of subsurface fatigue failure leading to the delamination. Surface chemistry and surface roughness of implant materials can have a significant influence on tissue damage, even when using materials and roughness values that satisfy regulatory requirements.

Porous titanium bases for osteochondral tissue engineering.

Tissue engineering of osteochondral grafts may offer a cell-based alternative to native allografts, which are in short supply. Previous studies promote the fabrication of grafts consisting of a viable cell-seeded hydrogel integrated atop a porous, bone-like metal. Advantages of the manufacturing process have led to the evaluation of porous titanium as the bone-like base material. Here, porous titanium was shown to support the growth of cartilage to produce native levels of Young's modulus, using a clinically relevant cell source. Mechanical and biochemical properties were similar or higher for the osteochondral constructs compared to chondral-only controls. Further investigation into the mechanical influence of the base on the composite material suggests that underlying pores may decrease interstitial fluid pressurization and applied strains, which may be overcome by alterations to the base structure. Future studies aim to optimize titanium-based tissue engineered osteochondral constructs to best match the structural architecture and strength of native grafts. STATEMENT OF SIGNIFICANCE: The studies described in this manuscript follow up on previous studies from our lab pertaining to the fabrication of osteochondral grafts that consist of a bone-like porous metal and a chondrocyte-seeded hydrogel. Here, tissue engineered osteochondral grafts were cultured to native stiffness using adult chondrocytes, a clinically relevant cell source, and a porous titanium base, a material currently used in clinical implants. This porous titanium is manufactured via selective laser melting, offering the advantages of precise control over shape, pore size, and orientation. Additionally, this manuscript describes the mechanical influence of the porous base, which may have applicability to porous bases derived from other materials.

Functional tissue engineering of chondral and osteochondral constructs.

Due to the prevalence of osteoarthritis (OA) and damage to articular cartilage, coupled with the poor intrinsic healing capacity of this avascular connective tissue, there is a great demand for an articular cartilage substitute. As the bearing material of diarthrodial joints, articular cartilage has remarkable functional properties that have been difficult to reproduce in tissue-engineered constructs. We have previously demonstrated that by using a functional tissue engineering approach that incorporates mechanical loading into the long-term culture environment, one can enhance the development of mechanical properties in chondrocyte-seeded agarose constructs. As these gel constructs begin to achieve material properties similar to that of the native tissue, however, new challenges arise, including integration of the construct with the underlying native bone. To address this issue, we have developed a technique for producing gel constructs integrated into an underlying bony substrate. These osteochondral constructs develop cartilage-like extracellular matrix and material properties over time in free swelling culture. In this study, as a preliminary to loading such osteochondral constructs, finite element modeling (FEM) was used to predict the spatial and temporal stress, strain, and fluid flow fields within constructs subjected to dynamic deformational loading. The results of these models suggest that while chondral ("gel alone") constructs see a largely homogenous field of mechanical signals, osteochondral ("gel bone") constructs see a largely inhomogeneous distribution of mechanical signals. Such inhomogeneity in the mechanical environment may aid in the development of inhomogeneity in the engineered osteochondral constructs. Together with experimental observations, we anticipate that such modeling efforts will provide direction for our efforts aimed at the optimization of applied physical forces for the functional tissue engineering of an osteochondral articular cartilage substitute.

Magnetic hydroxyapatite bone substitutes to enhance tissue regeneration: evaluation in vitro using osteoblast-like cells and in vivo in a bone defect.

In case of degenerative disease or lesion, bone tissue replacement and regeneration is an important clinical goal. In particular, nowadays, critical size defects rely on the engineering of scaffolds that are 3D structural supports, allowing cellular infiltration and subsequent integration with the native tissue. Several ceramic hydroxyapatite (HA) scaffolds with high porosity and good osteointegration have been developed in the past few decades but they have not solved completely the problems related to bone defects. In the present study we have developed a novel porous ceramic composite made of HA that incorporates magnetite at three different ratios: HA/Mgn 95/5, HA/Mgn 90/10 and HA/Mgn 50/50. The scaffolds, consolidated by sintering at high temperature in a controlled atmosphere, have been analysed in vitro using human osteoblast-like cells. Results indicate high biocompatibility, similar to a commercially available HA bone graft, with no negative effects arising from the presence of magnetite or by the use of a static magnetic field. HA/Mgn 90/10 was shown to enhance cell proliferation at the early stage. Moreover, it has been implanted in vivo in a critical size lesion of the rabbit condyle and a good level of histocompatibility was observed. Such results identify this scaffold as particularly relevant for bone tissue regeneration and open new perspectives for the application of a magnetic field in a clinical setting of bone replacement, either for magnetic scaffold fixation or magnetic drug delivery.

Trimethylamine N-oxide as a media supplement for cartilage tissue engineering.

Supplements added to the culture media (e.g., growth factors and dexamethasone) have been successful in improving mechanical and biochemical properties of engineered cartilage towards native values. Trimethylamine N-oxide (TMAO), a natural osmolyte found in shark cartilage, is thought to induce protein folding, and counteracts the destabilizing effect of the high concentrations of urea stored by sharks. The objective of this study was to investigate the use of TMAO as a media supplement for promoting growth of functional engineered cartilage in culture. In the first study, TMAO was added to the culture media for the first 14 days in culture and concentrations of 0-200 mM were evaluated. In the second study, TMAO was supplemented to the culture media following chondroitinase ABC digestion, which has been previously shown to mediate an increased collagen content in engineered cartilage. A dose-dependent response was observed with improved mechanical and biochemical properties for engineered constructs cultured with TMAO at concentrations of 5-100 mM. The Young's modulus of digested constructs cultured in TMAO was 2× greater than digested constructs cultured in the control medium and recovered to undigested control levels by day 42. In conclusion, these initial studies with TMAO as a media supplement show promise for improving the compressive mechanical properties, increasing extracellular matrix production, and increasing the recovery time following chABC digestion.

Effects of hypertonic (NaCl) two-dimensional and three-dimensional culture conditions on the properties of cartilage tissue engineered from an expanded mature bovine chondrocyte source.

Clinically relevant mature cartilage cells (chondrocytes) present challenges for use in cartilage tissue engineering applications, given their low capacity for cell division and tissue production. Since the in situ environment of chondrocytes is hypertonic relative to standard culture medium conditions, in this study we tested the hypothesis that using culture medium of a hypertonic, more physiologic osmolarity during both two-dimensional (2D) expansion of mature bovine chondrocytes (MBCs) and their subsequent encapsulation culture in three-dimensional (3D) agarose hydrogel constructs produces improved engineered tissue construct mechanical and biochemical properties. Results demonstrate that 2D expansion of MBCs in hypertonic (NaCl) medium before encapsulation yielded improved construct mechanical properties. However, 3D encapsulation culture of cells in hypertonic (NaCl) medium yielded poorer construct mechanical properties. Osmolarity-related differences in construct biochemical content and organization may have contributed to differences in mechanical properties, as construct glycosaminoglycan content correlated moderately with construct mechanical properties, and construct collagen distribution varied between 3D osmotic culture groups. Results of this study suggest that application of hypertonic (NaCl) medium during 2D mature chondrocyte expansion, but not 3D encapsulated chondrocyte culture, may serve as a convenient and inexpensive method for improving mechanical properties of expanded cell-seeded constructs.

Transient supplementation of anabolic growth factors rapidly stimulates matrix synthesis in engineered cartilage.

The purpose of the presented work is to examine the response of engineered cartilage to a transient, 2-week application of anabolic growth factors compared to continuous exposure in in vitro culture. Immature bovine chondrocytes were suspended in agarose hydrogel and cultured for 28 days (Study 1) or 42 days (Study 2) in chondrogenic media with TGF-ß1, TGF-ß3, or IGF-I either added for only the first 14 days in culture or added to the media for the entire study period. In both studies, there were no statistical differences in tissue mechanical or biochemical properties between the growth factors on day 14. In Study 1, growth factor removal led to a significant and drastic increase in Young's modulus and glycosaminoglycans content compared to continuously exposed controls on day 28. In Study 2, both TGF-ß1 and ß3 led to significantly higher mechanical properties and collagen content vs. IGF-I on day 42. These results indicate that the rapid rise in tissue properties (previously observed with TGF-ß3 only) is not dependent on the type but rather the temporal application of the anabolic growth factor. These findings shed light on possible techniques to rapidly develop engineered cartilage tissue for the future treatment of osteoarthritis.

Bioactive stratified polymer ceramic-hydrogel scaffold for integrative osteochondral repair.

Due to the intrinsically poor repair potential of articular cartilage, injuries to this soft tissue do not heal and require clinical intervention. Tissue engineered osteochondral grafts offer a promising alternative for cartilage repair. The functionality and integration potential of these grafts can be further improved by the regeneration of a stable calcified cartilage interface. This study focuses on the design and optimization of a stratified osteochondral graft with biomimetic multi-tissue regions, including a pre-designed and pre-integrated interface region. Specifically, the scaffold based on agarose hydrogel and composite microspheres of polylactide-co-glycolide (PLGA) and 45S5 bioactive glass (BG) was fabricated and optimized for chondrocyte density and microsphere composition. It was observed that the stratified scaffold supported the region-specific co-culture of chondrocytes and osteoblasts which can lead to the production of three distinct yet continuous regions of cartilage, calcified cartilage and bone-like matrices. Moreover, higher cell density enhanced chondrogenesis and improved graft mechanical property over time. The PLGA-BG phase promoted chondrocyte mineralization potential and is required for the formation of a calcified interface and bone regions on the osteochondral graft. These results demonstrate the potential of the stratified scaffold for integrative cartilage repair and future studies will focus on scaffold optimization and in vivo evaluations.

Passaged adult chondrocytes can form engineered cartilage with functional mechanical properties: a canine model.

It was hypothesized that previously optimized serum-free culture conditions for juvenile bovine chondrocytes could be adapted to generate engineered cartilage with physiologic mechanical properties in a preclinical, adult canine model. Primary or passaged (using growth factors) adult chondrocytes from three adult dogs were encapsulated in agarose, and cultured in serum-free media with transforming growth factor-beta3. After 28 days in culture, engineered cartilage formed by primary chondrocytes exhibited only small increases in glycosaminoglycan content. However, all passaged chondrocytes on day 28 elaborated a cartilage matrix with compressive properties and glycosaminoglycan content in the range of native adult canine cartilage values. A preliminary biocompatibility study utilizing chondral and osteochondral constructs showed no gross or histological signs of rejection, with all implanted constructs showing excellent integration with surrounding cartilage and subchondral bone. This study demonstrates that adult canine chondrocytes can form a mechanically functional, biocompatible engineered cartilage tissue under optimized culture conditions. The encouraging findings of this work highlight the potential for tissue engineering strategies using adult chondrocytes in the clinical treatment of cartilage defects.

One breath closer to making engineered tissues a clinical reality.

Reported recently in Lancet, Macchiarini and colleagues (2008) implanted a living tissue-engineered airway in a female patient. The restoration of the patient's quality of life testifies to this successful translation of benchtop to bedside studies and provides promise for the application of regenerative medicine strategies to other clinical disorders.

Zonal chondrocytes seeded in a layered agarose hydrogel create engineered cartilage with depth-dependent cellular and mechanical inhomogeneity.

We hypothesized that zonal populations of chondrocytes seeded into a bilayered scaffold with initially prescribed depth-varying, compressive material properties will lead to a biomimetic cartilage tissue construct with depth-dependent cellular and compressive mechanical inhomogeneity similar to that of the native tissue. Superficial zone chondrocytes (SZCs) and middle/deep zone chondrocytes (MDZCs) were isolated and encapsulated with 2% or 3% agarose to form single-layered constructs of 2% SZC, 3% SZC, 2% MDZC; bilayered constructs of 2% SZC/2% MDZC and 3% SZC/2% MDZC; and 2% mixed chondrocyte controls. For SZCs on day 42, increased glycosaminoglycan (GAG) and collagen was found with increased agarose concentration and when layered with MDZCs. Superficial zone protein increased with agarose concentration in bilayered constructs. For MDZCs, increased GAG content and regulation of cell proliferation was observed when layered with SZCs. Bilayered constructs possessed a depth-dependent compressive modulus qualitatively similar to that of native articular cartilage, whereas controls showed a U-shaped profile with stiffer peripheral edges and softer middle region. This study is the first to create an engineered cartilage tissue with depth-varying cellular as well as mechanical inhomogeneity. Future studies will determine if replicating inhomogeneity is advantageous in clinical applications of tissue engineered cartilage.

Genipin enhances the mechanical properties of tissue-engineered cartilage and protects against inflammatory degradation when used as a medium supplement.

Genipin is a naturally-derived biocompatible cross-linking agent commonly used to generate three dimensional tissue-engineered scaffolds or to fix biologically derived scaffolds prior to implantation. Here we propose a novel use for genipin as a long-term culture medium supplement to promote cross-linking of de novo cell products that are produced in engineered cartilage. We hypothesize that the application of genipin will stabilize the extracellular matrix components and increase the mechanical properties of developing engineered cartilage. Chondrocytes encapsulated in agarose hydrogel (a neutrally charged polysaccharide scaffold that is unaffected by genipin cross-linking) were cultured in a chemically-defined growth medium that was supplemented with varying concentrations of genipin (22 microM, 220 microM, 2200 microM) for various durations (continuous or intermittent). Tissues developed significantly higher mechanical properties (+28% dynamic modulus and +20% Young's modulus) by day 42 with genipin treatment compared to untreated controls. These increases were not immediate, but presented over culture time after genipin treatment. The genipin treated groups were also more resistant to cytokine-induced degradation with interleukin-1alpha; maintaining an E(Y) (+218%), G* (+390%) and glycosaminoglycan (GAG) content (+477%) over genipin-untreated constructs subjected to interleukin. We hypothesize two mechanisms through which the physical enhancement of tissue properties may be fostered: (1) by cross-link mediated reorganization and enhanced retention of cell-elaborated extracellular matrix components, and (2) through reduction of the loss of extracellular matrix components by increasing their resilience to catabolic degradation. These studies demonstrate a potential use of genipin as a medium supplement to develop enhanced engineered cartilage.

Intracellular calcium waves in bone cell networks under single cell nanoindentation.

In this study, bone cells were successfully cultured into a micropatterned network with dimensions close to that of in vivo osteocyte networks using microcontact printing and self-assembled monolyers (SAMs). The optimal geometric parameters for the formation of these networks were determined in terms of circle diameters and line widths. Bone cells patterned in these networks were also able to form gap junctions with each other, shown by immunofluorescent staining for the gap junction protein connexin 43, as well as the transfer of gap-junction permeable calcein-AM dye. We have demonstrated for the first time, that the intracellular calcium response of a single bone cell indented in this bone cell network, can be transmitted to neighboring bone cells through multiple calcium waves. Furthermore, the propagation of these calcium waves was diminished with increased cell separation distance. Thus, this study provides new experimental data that support the idea of osteocyte network memory of mechanical loading similar to memory in neural networks.

Direct measurement of osmotic pressure of glycosaminoglycan solutions by membrane osmometry at room temperature.

Articular cartilage is a hydrated soft tissue composed of negatively charged proteoglycans fixed within a collagen matrix. This charge gradient causes the tissue to imbibe water and swell, creating a net osmotic pressure that enhances the tissue's ability to bear load. In this study we designed and utilized an apparatus for directly measuring the osmotic pressure of chondroitin sulfate, the primary glycosaminoglycan found in articular cartilage, in solution with varying bathing ionic strength (0.015 M, 0.15 M, 0.5 M, 1 M, and 2 M NaCl) at room temperature. The osmotic pressure (pi) was found to increase nonlinearly with increasing chondroitin sulfate concentration and decreasing NaCl ionic bath environment. Above 1 M NaCl, pi changes negligibly with further increases in salt concentration, suggesting that Donnan osmotic pressure is negligible above this threshold, and the resulting pressure is attributed to configurational entropy. Results of the current study were also used to estimate the contribution of osmotic pressure to the stiffness of cartilage based on theoretical and experimental considerations. Our findings indicate that the osmotic pressure resulting from configurational entropy is much smaller in cartilage (based on an earlier study on bovine articular cartilage) than in free solution. The rate of change of osmotic pressure with compressive strain is found to contribute approximately one-third of the compressive modulus (H(A)(eff)) of cartilage (Pi approximately H(A)(eff)/3), with the balance contributed by the intrinsic structural modulus of the solid matrix (i.e., H(A) approximately 2H(A)(eff)/3). A strong dependence of this intrinsic modulus on salt concentration was found; therefore, it appears that proteoglycans contribute structurally to the magnitude of H(A), in a manner independent of osmotic pressure.