A Trabecular Bone Explant Model of Osteocyte–Osteoblast Co-Culture for Bone Mechanobiology

The osteocyte network is recognized as the major mechanical sensor in the bone remodeling process, and osteocyte-osteoblast communication acts as an important mediator in the coordination of bone formation and turnover. In this study, we developed a novel 3D trabecular bone explant co-culture model that allows live osteocytes situated in their native extracellular matrix environment to be interconnected with seeded osteoblasts on the bone surface. Using a low-level medium perfusion system, the viability of in situ osteocytes in bone explants was maintained for up to 4 weeks, and functional gap junction intercellular communication (GJIC) was successfully established between osteocytes and seeded primary osteoblasts. Using this novel co-culture model, the effects of dynamic deformational loading, GJIC, and prostaglandin E-2 (PGE(2)) release on functional bone adaptation were further investigated. The results showed that dynamical deformational loading can significantly increase the PGE(2) release by bone cells, bone formation, and the apparent elastic modulus of bone explants. However, the inhibition of gap junctions or the PGE(2) pathway dramatically attenuated the effects of mechanical loading. This 3D trabecular bone explant co-culture model has great potential to fill in the critical gap in knowledge regarding the role of osteocytes as a mechano-sensor and how osteocytes transmit signals to regulate osteoblasts function and skeletal integrity as reflected in its mechanical properties.

Fluid flow induced calcium response in bone cell network

In our previous work, bone cell networks with controlled spacing and functional intercellular gap junctions had been successfully established by using microcontact printing and self assembled monolayers technologies [Guo, X. E., E. Takai, X. Jiang, Q. Xu, G. M. Whitesides, J. T. Yardley, C. T. Hung, E. M. Chow, T. Hantschel, and K. D. Costa. Mol. Cell. Biomech. 3:95-107, 2006]. The present study investigated the calcium response and the underlying signaling pathways in patterned bone cell networks exposed to a steady fluid flow. The glass slides with cell networks were separated into eight groups for treatment with specific pharmacological agents that inhibit pathways significant in bone cell calcium signaling. The calcium transients of the network were recorded and quantitatively evaluated with a set of network parameters. The results showed that 18 alpha-GA (gap junction blocker), suramin (ATP inhibitor), and thapsigargin (depleting intracellular calcium stores) significantly reduced the occurrence of multiple calcium peaks, which were visually obvious in the untreated group. The number of responsive peaks also decreased slightly yet significantly when either the COX-2/PGE(2) or the NOS/nitric oxide pathway was disrupted. Different from all other groups, cells treated with 18 alpha-GA maintained a high concentration of intracellular calcium following the first peak. In the absence of calcium in the culture medium, the intracellular calcium concentration decreased slowly with fluid flow without any calcium transients observed. These findings have identified important factors in the flow mediated calcium signaling of bone cells within a patterned network.

Parametric Analysis for Monitoring 2D Kinetics of Receptor-Ligand binding

Thermal fluctuation approach is widely used to monitor association kinetics of surface-bound receptor-ligand interactions. Various protocols such as sliding standard deviation (SD) analysis (SSA) and Page's test analysis (PTA) have been used to estimate two-dimensional (2D) kinetic rates from the time course of displacement of molecular carrier. In the current work, we compared the estimations from both SSA and modified PTA using measured data from an optical trap assay and simulated data from a random number generator. Our results indicated that both SSA and PTA were reliable in estimating 2D kinetic rates. Parametric analysis also demonstrated that such the estimations were sensitive to parameters such as sampling rate, sliding window size, and threshold. These results furthered the understandings in quantifying the biophysics of receptor-ligand interactions.

Membrane Deformability and Membrane Tension of Single Isolated Mitochondria

Mitochondria dynamics is crucial to many biological processes such as mitochondria fusion and fission, which is highly correlated to the mechanics of single mitochondria. However, the mechanobiological coupling of mitochondria has been poorly understood. Here membrane deformability and membrane tension of individual mitochondria isolated from MtDsRed labeled human embryonic T-Rex-293 kidney cells were measured using a micropipette aspiration assay. The results demonstrated that membrane deformation of isolated mitochondria exhibited an elastic transition phase followed by an equilibrium phase, and mitochondrial membrane tension was proportional to the area compressibility. It was also indicated that mitochondrial membrane deformability was significantly affected by physical chemical factors such as osmotic pressure or pH value, and was further correlated to mitochondrial functionality in different respiratory states and Ca2+ regulation. These findings provide a new insight into understanding the mechanical regulation of mitochondrial physiology.

O uso de células-tronco na regeneração dos tecidos dentários e periodonto

Os estudos abordando a regeneração dos tecidos dentários ganharam uma nova perspectiva com a utilização das células-tronco. E novas perspectivas têm surgido com a bioengenharia tecidual e as terapias periodontais e pulpares regeneradoras. O objetivo deste trabalho foi desenvolver o modelo experimental de autotransplante em ratos visando compará-lo à técnica de reimplante e estudar a capacidade terapêutica das células da medula óssea em diferentes biomateriais utilizados como matriz para a terapia de células-tronco no reparo dos tecidos dentais. Foram utilizados 23 ratos Wistar divididos em grupos de 1, 3, 15 e 60 dias para as técnicas de reimplante e autotransplante. Os grupos com injeção de células-tronco (CT) foram: (1) grupo de 3 dias, combinado à técnica de reimplante; (2) grupo de 15 dias com ambas as técnicas. Blocos contendo os três dentes molares superiores de cada lado dos ratos foram removidos, feitas radiografias periapicais e as peças foram processadas para inclusão em parafina. Foram avaliadas a espessura do ligamento periodontal (LPD) comparada entre os diferentes grupos e a morfologia celular e matriz extracelular relacionadas à superfície radicular, ao osso alveolar e à porção média do LPD, além das células da polpa dental de cada grupo. As células isoladas a partir da medula-óssea foram incubadas por 24h, 48h, e 72h em placas de cultura contendo membranas de colágeno bovino tipo I - CollaTape (Integra LifeSciences Corporation, Plainsboro, NJ, USA), enxerto ósseo - Extra Graft XG-13 (Silvestre Labs Quimica e Farmaceutica LTDA, RJ, Brazil) ou um dente molar de rato. Os espécimes foram observados em um microscópio invertido para contagem de células e processadas para observação no microscópio eletrônico de varredura (MEV). Os grupos de 1 e 3 dias apresentaram medidas de LPD significativamente maiores para a técnica de autotransplante quando comparadas ao reimplante. O grupo de 3 dias com CT não apresentou alterações pulpares significativas, diferente do controle (sem CT) O grupo de 15 dias com CT apresentou as mesmas características histológicas do grupo sem injeção de CT. A observação ao MEV dos biomateriais revelou que as células apresentaram pouca adesão e proliferação no enxerto ósseo e no cemento dentário quando comparados à membrana colágena. A técnica de reimplante associada à injeção de células-tronco sugere alguma influência da terapia com as células-tronco sobre a polpa. As distâncias aumentadas no LPD com a técnica de autotransplante podem não influenciar tanto o sucesso da técnica. As células mesenquimais da medula óssea possuem grande potencial para colonizarem a membrana colágena CollaTape que mostrou vantagens sobre o enxerto ósseo Extra Graft XG-13 como biomaterial para a aderência e a proliferação de células mononucleares da medula óssea, permitindo a diferenciação destas células.

Poroviscoelastic characterization of particle-reinforced gelatin gels using indentation and homogenization.

Hydrogels are promising materials for bioengineering applications, and are good model materials for the study of hydrated biological tissues. As these materials often have a structural function, the measurement of their mechanical properties is of fundamental importance. In the present study gelatin gels reinforced with ceramic microspheres are produced and their poroviscoelastic response in spherical indentation is studied. The constitutive responses of unreinforced gels are determined using inverse finite element modeling in combination with analytical estimates of material parameters. The behavior of composite gels is assessed by both analytical and numerical homogenization. The results of the identification of the constitutive parameters of unreinforced gels show that it is possible to obtain representative poroviscoelastic parameters by spherical indentation without the need for additional mechanical tests. The agreement between experimental results on composite gelatin and the predictions from homogenization modeling show that the adopted modeling tools are capable of providing estimates of the poroviscoelastic response of particle-reinforced hydrogels.

Biomimicking of natural cellular materials

The role that microstructure plays in the mechanical efficiency of natural cellular materials is examined here. The focus of this study is on elastic behaviour. Two natural materials with microstructures resistant to local bucking: plant stems and animal quills have also been examined.

Variability of nanoindentation responses of bone and artificial bone-like composites

Nanoindentation is a popular technique for measuring the intrinsic mechanical response of bone and has been used to measure a single-valued elastic modulus. However, bone is a composite material with 20-80 nm hydroxyapatite plates embedded in a collagen matrix, and modern instrumentation allows for measurements at these small length scales. The present study examines the indentation response of bone and artificial gelatin-apatite nanocomposite materials across three orders of magnitude of lengthscale, from nanometers to micrometers, to isolate the composite phase contributions to the overall response. The load-displacement responses were variable and deviated from the quadratic response of homogeneous materials at small depths. The distribution of apparent elastic modulus values narrowed substantially with increasing indentation load. Indentation of particulate nanocomposites was simulated using finite element analysis. Modeling results replicated the convergence in effective modulus seen in the experiments. It appears that the apatite particles are acting as the continuous ("matrix") phase in bone and nanocomposites. Copyright © 2004 by ASME.

Mineralization and nanomechanical properties in Articular Calcified Cartilage (ACC)

The contribution of the relative volumes of mineral and collagen to the nanomechanical behavior of articular calcified cartilage is explored using nanoindentation, quantitative backscattered electron imaging, and finite element analysis. Elastic modulus generally increases with mineral volume fraction. In highly mineralized tissues, the mineral occupation of water space significantly increases modulus with addition of little mineral. Mineral and organic phases were modeled using Hashin-Shtrikman composite bounds, calculated as a function of mineral volume fraction. Modulus values fall between the Hashin-Shtrikman bounds, indicating some intermediate degree of mineral phase connectivity. Such connectivity in ACC is greater than that achieved in bone and results from uniform collagen orientation and large volume of water space available for mineral occupation.