Autophagy Is Possibly Involved in Osteoblast Responses to Mechanical Loadings.

Both mechanical loading and autophagy play important roles in regulating bone growth and remodeling, but the relationship between the two remains unclear. In this study, we examined bone structure with micro-CT imaging and measured bone mechanical properties with three-point bending experiments using bones from wild-type (WT) mice and conditional knockout (cKO) mice with Atg7 deletion in their osteoblasts. We found that the knockout mice had significantly less bone volume, bone thickness, bone ultimate breaking force, and bone stiffness compared to wild-type mice. Additionally, bone marrow cells from knockout mice had reduced differentiation and mineralization capacities in terms of alkaline phosphatase and calcium secretion, as well as Runx2 and osteopontin expression. Knockout mice also had significantly less relative bone formation rate due to mechanical loading. Furthermore, we found that the osteoblasts from wild-type mice had stronger responses to mechanical stimulation compared to autophagy-deficient osteoblasts from knockout mice. When inhibiting autophagy with 3 MA in wild-type osteoblasts, we found similar results as we did in autophagy-deficient osteoblasts. We also found that mechanical loading-induced ATP release is able to regulate ERK1/2, Runx2, alkaline phosphatase, and osteopontin activities. These results suggest that the ATP pathway may play an important role in the possible involvement of autophagy in osteoblast mechanobiology.

Combining 3D skeleton data and deep convolutional neural network for balance assessment during walking.

Introduction: Balance impairment is an important indicator to a variety of diseases. Early detection of balance impairment enables doctors to provide timely treatments to patients, thus reduce their fall risk and prevent related disease progression. Currently, balance abilities are usually assessed by balance scales, which depend heavily on the subjective judgement of assessors. Methods: To address this issue, we specifically designed a method combining 3D skeleton data and deep convolutional neural network (DCNN) for automated balance abilities assessment during walking. A 3D skeleton dataset with three standardized balance ability levels were collected and used to establish the proposed method. To obtain better performance, different skeleton-node selections and different DCNN hyperparameters setting were compared. Leave-one-subject-out-cross-validation was used in training and validation of the networks. Results and Discussion: Results showed that the proposed deep learning method was able to achieve 93.33% accuracy, 94.44% precision and 94.46% F1 score, which outperformed four other commonly used machine learning methods and CNN-based methods. We also found that data from body trunk and lower limbs are the most important while data from upper limbs may reduce model accuracy. To further validate the performance of the proposed method, we migrated and applied a state-of-the-art posture classification method to the walking balance ability assessment task. Results showed that the proposed DCNN model improved the accuracy of walking balance ability assessment. Layer-wise Relevance Propagation (LRP) was used to interpret the output of the proposed DCNN model. Our results suggest that DCNN classifier is a fast and accurate method for balance assessment during walking.

ICTs and interventions in telerehabilitation and their effects on stroke recovery.

Telerehabilitation (TR) is a new model to provide rehabilitation services to stroke survivors. It is a promising approach to deliver mainstream interventions for movement, cognitive, speech and language, and other disorders. TR has two major components: information and communication technologies (ICTs) and stroke interventions. ICTs provide a platform on which interventions are delivered and subsequently result in stroke recovery. In this mini-review, we went over features of ICTs that facilitate TR, as well as stroke interventions that can be delivered via TR platforms. Then, we reviewed the effects of TR on various stroke disorders. In most studies, TR is a feasible and effective solution in delivering interventions to patients. It is not inferior to usual care and in-clinic therapy with matching dose and intensity. With new technologies, TR may result in better outcomes than usual care for some disorders. One the other hand, TR also have many limitations that could lead to worse outcomes than traditional rehabilitation. In the end, we discussed major concerns and possible solutions related to TR, and also discussed potential directions for TR development.

The role of smart polymeric biomaterials in bone regeneration: a review.

Addressing critical bone defects necessitates innovative solutions beyond traditional methods, which are constrained by issues such as immune rejection and donor scarcity. Smart polymeric biomaterials that respond to external stimuli have emerged as a promising alternative, fostering endogenous bone regeneration. Light-responsive polymers, employed in 3D-printed scaffolds and photothermal therapies, enhance antibacterial efficiency and bone repair. Thermo-responsive biomaterials show promise in controlled bioactive agent release, stimulating osteocyte differentiation and bone regeneration. Further, the integration of conductive elements into polymers improves electrical signal transmission, influencing cellular behavior positively. Innovations include advanced 3D-printed poly (l-lactic acid) scaffolds, polyurethane foam scaffolds promoting cell differentiation, and responsive polymeric biomaterials for osteogenic and antibacterial drug delivery. Other developments focus on enzyme-responsive and redox-responsive polymers, which offer potential for bone regeneration and combat infection. Biomaterials responsive to mechanical, magnetic, and acoustic stimuli also show potential in bone regeneration, including mechanically-responsive polymers, magnetic-responsive biomaterials with superparamagnetic iron oxide nanoparticles, and acoustic-responsive biomaterials. In conclusion, smart biopolymers are reshaping scaffold design and bone regeneration strategies. However, understanding their advantages and limitations is vital, indicating the need for continued exploratory research.

Light responsive hydrogels for controlled drug delivery.

Light is an easy acquired, effective and non-invasive external stimulus with great flexibility and focusability. Thus, light responsive hydrogels are of particular interests to researchers in developing accurate and controlled drug delivery systems. Light responsive hydrogels are obtained by incorporating photosensitive moieties into their polymeric structures. Drug release can be realized through three major mechanisms: photoisomerization, photochemical reaction and photothermal reaction. Recent advances in material science have resulted in great development of photosensitizers, such as rare metal nanostructures and black phosphorus nanoparticles, in order to respond to a variety of light sources. Hydrogels incorporated with photosensitizers are crucial for clinical applications, and the use of ultraviolet and near-infrared light as well as up-conversion nanoparticles has greatly increased the therapeutic effects. Existing light responsive drug delivery systems have been utilized in delivering drugs, proteins and genes for chemotherapy, immunotherapy, photodynamic therapy, gene therapy, wound healing and other applications. Principles associated with site-specific targeting, metabolism, and toxicity are used to optimize efficacy and safety, and to improve patient compliance and convenience. In view of the importance of this field, we review current development, challenges and future perspectives of light responsive hydrogels for controlled drug delivery.

Effect of microtopography on osseointegration of implantable biomaterials and its modification strategies.

Orthopedic implants are widely used for the treatment of bone defects caused by injury, infection, tumor and congenital diseases. However, poor osseointegration and implant failures still occur frequently due to the lack of direct contact between the implant and the bone. In order to improve the biointegration of implants with the host bone, surface modification is of particular interest and requirement in the development of implant materials. Implant surfaces that mimic the inherent surface roughness and hydrophilicity of native bone have been shown to provide osteogenic cells with topographic cues to promote tissue regeneration and new bone formation. A growing number of studies have shown that cell attachment, proliferation and differentiation are sensitive to these implant surface microtopography. This review is to provide a summary of the latest science of surface modified bone implants, focusing on how surface microtopography modulates osteoblast differentiation in vitro and osseointegration in vivo, signaling pathways in the process and types of surface modifications. The aim is to systematically provide comprehensive reference information for better fabrication of orthopedic implants.

Effects of membrane cholesterol depletion and GPI-anchored protein reduction on osteoblastic mechanotransduction.

We previously demonstrated that oscillatory fluid flow activates MC3T3-E1 osteoblastic cell calcium signaling pathways via a mechanism involving ATP releases and P2Y(2) puringeric receptors. However, the molecular mechanisms by which fluid flow initiates cellular responses are still unclear. Accumulating evidence suggests that lipid rafts, one of the important membrane structural components, may play an important role in transducing extracellular fluid shear stress to intracellular responses. Due to the limitations of current techniques, there is no direct approach to study the role of lipid rafts in transmitting fluid shear stress. In this study, we targeted two important membrane components associated with lipid rafts, cholesterol, and glycosylphosphatidylinositol-anchored proteins (GPI-anchored proteins), to disrupt the integrity of cell membrane structures. We first demonstrated that membrane cholesterol depletion with the treatment of methyl-ß-cyclodextrin inhibits oscillatory fluid flow induced intracellular calcium mobilization and ERK1/2 phosphorylation in MC3T3-E1 osteoblastic cells. Secondly, we used a novel approach to decrease the levels of GPI-anchored proteins on cell membranes by overexpressing glycosylphosphatidylinositol-specific phospholipase D in MC3T3-E1 osteoblastic cells. This resulted in significant inhibition of intracellular calcium mobilization and ERK1/2 phosphorylation in response to oscillatory fluid flow. Finally, we demonstrated that cholesterol depletion inhibited oscillatory fluid flow induced ATP releases, which were responsible for the activation of calcium signaling pathways in MC3T3-E1 osteoblastic cells. Our findings suggest that cholesterol and GPI-anchored proteins, two membrane structural components related to lipid rafts, may play an important role in osteoblastic cell mechanotransduction.

Purinergic Signaling Mediates PTH and Fluid Flow-Induced Osteoblast Proliferation.

Both parathyroid hormone (PTH) and mechanical signals are able to regulate bone growth and regeneration. They also can work synergistically to regulate osteoblast proliferation, but little is known about the mechanisms how PTH and mechanical signals interact with each other during this process. In this study, we investigated responses of MC3T3-E1 osteoblasts to PTH and oscillatory fluid flow. We found that osteoblasts are more sensitive to mechanical signals in the presence of PTH according to ERK1/2 phosphorylation, ATP release, CREB phosphorylation, and cell proliferation. PTH may also reduce the osteoblast refractory period after desensitization due to mechanical signals. We further found that the synergistic responses of osteoblasts to fluid flow or ATP with PTH had similar patterns, suggesting that synergy between fluid flow and PTH may be through the ATP pathway. After we inhibited ATP effects using apyrase in osteoblasts, their synergistic responses to mechanical stimulation and PTH were also inhibited. Additionally, knocking down P2Y2 purinergic receptors can significantly attenuate osteoblast synergistic responses to mechanical stimulation and PTH in terms of ERK1/2 phosphorylation, CREB phosphorylation, and cell proliferation. Thus, our results suggest that PTH enhances mechanosensitivity of osteoblasts via a mechanism involving ATP and P2Y2 purinergic receptors.

GIT1 utilizes a focal adhesion targeting-homology domain to bind paxillin.

The GIT proteins, GIT1 and GIT2, are GTPase-activating proteins for the ADP-ribosylation factor family of small GTP-binding proteins, but also serve as adaptors to link signaling proteins to distinct cellular locations. One role for GIT proteins is to link the PIX family of Rho guanine nucleotide exchange factors and their binding partners, the p21-activated protein kinases, to remodeling focal adhesions by interacting with the focal adhesion adaptor protein paxillin. We here identified the C-terminal domain of GIT1 responsible for paxillin binding. Combining structural and mutational analyses, we show that this region folds into an anti-parallel four-helix domain highly reminiscent to the focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK). Our results suggest that the GIT1 FAT-homology (FAH) domain and FAT bind the paxillin LD4 motif quite similarly. Since only a small fraction of GIT1 is bound to paxillin under normal conditions, regulation of paxillin binding was explored. Although paxillin binding to the FAT domain of FAK is regulated by tyrosine phosphorylation within this domain, we find that tyrosine phosphorylation of the FAH domain GIT1 is not involved in regulating binding to paxillin. Instead, we find that mutations within the FAH domain may alter binding to paxillin that has been phosphorylated within the LD4 motif. Thus, despite apparent structural similarity in their FAT domains, GIT1 and FAK binding to paxillin is differentially regulated.

Osteogenesis-Related Behavior of MC3T3-E1 Cells on Substrates with Tunable Stiffness.

Osteogenic differentiation of cells has considerable clinical significance in bone defect treatment, and cell behavior is linked to extracellular matrix stiffness. This study aimed to determine how matrix stiffness affects cell morphology and subsequently regulates the osteogenic phenotype of osteogenesis precursor cells. Four PDMS substrates were prepared with stiffness corresponding to the elastic modulus ranging from 0.6 MPa to 2.7 MPa by altering the Sylgard 527 and Sylgard 184 concentrations. MC3T3-E1 cells were cultured on the matrices. Cell morphology, vinculin expression, and key osteogenic markers, Col I, OCN, OPN, and calcium nodule, were examined. The activity and expression level of Yes-associated protein (YAP) were evaluated. Results showed that cell spreading exhibited no correlation with the stiffness of matrix designed in this paper, but substratum stiffness did modulate MC3T3-E1 osteogenic differentiation. Col I, OPN, and OCN proteins were significantly increased in cells cultured on soft matrices compared with stiff matrices. Additionally, cells cultured on the 1:3 ratio matrices had more nodules than those on other matrices. Accordingly, cells on substrates with low stiffness showed enhanced expression of the osteogenic markers. Meanwhile, YAP expression was downregulated on soft substrates although the subcellular location was not affected. Our results provide evidence that matrix stiffness (elastic modulus ranging from 0.6 MPa to 2.7 MPa) affects the osteogenic differentiation of MC3T3-E1, but it is not that "the stiffer, the better" as showed in some of the previous studies. The optimal substrate stiffness may exist to promote osteoblast differentiation. Cell differentiation triggered by the changes in substrate stiffness may be independent of the YAP signal. This study has important implications for biomaterial design and stem cell-based tissue engineering.

The GIT/PIX complex: an oligomeric assembly of GIT family ARF GTPase-activating proteins and PIX family Rac1/Cdc42 guanine nucleotide exchange factors.

GIT proteins are GTPase-activating proteins (GAPs) for ADP-ribosylation factor (ARF) small GTP-binding proteins, and interact with the PIX family of Rac1/Cdc42 guanine nucleotide exchange factors. GIT and PIX transiently localize p21-activated protein kinases (PAKs) to remodeling focal adhesions through binding to paxillin. To understand the role of these interactions, the association of GIT and PIX proteins was examined in detail. Two separable binding interactions link GIT and PIX proteins, GIT and PIX proteins each dimerize and a beta-PIX fragment containing the GIT-binding region failed to inhibit the association of the GIT and PIX proteins. Endogenous GIT and PIX co-fractionate at a very high molecular size. Purified 6xHis-tagged beta-PIX from Sf9 cells co-expressing untagged GIT1 yields recombinant GIT1/beta-PIX complexes that have equal amounts of beta-PIX and GIT1 and co-fractionate at the same large size as native GIT/PIX complexes. Thus, GIT and PIX proteins are tightly associated as a multimeric nexus capable of linking together important signaling molecules, including PAKs.

The roles of P2Y2 purinergic receptors in osteoblasts and mechanotransduction.

We previously demonstrated, using osteoblastic MC3T3-E1 cells, that P2Y2 purinergic receptors are involved in osteoblast mechanotransduction. In this study, our objective was to further investigate, using a knockout mouse model, the roles of P2Y2 receptors in bone mechanobiology. We first examined bone structure with micro-CT and measured bone mechanical properties with three point bending experiments in both wild type mice and P2Y2 knockout mice. We found that bones from P2Y2 knockout mice have significantly decreased bone volume, bone thickness, bone stiffness and bone ultimate breaking force at 17 week old age. In order to elucidate the mechanisms by which P2Y2 receptors contribute to bone biology, we examined differentiation and mineralization of bone marrow cells from wild type and P2Y2 knockout mice. We found that P2Y2 receptor deficiency reduces the differentiation and mineralization of bone marrow cells. Next, we compared the response of primary osteoblasts, from both wild type and P2Y2 knockout mice, to ATP and mechanical stimulation (oscillatory fluid flow), and found that osteoblasts from wild type mice have a stronger response, in terms of ERK1/2 phosphorylation, to both ATP and fluid flow, relative to P2Y2 knockout mice. However, we did not detect any difference in ATP release in response to fluid flow between wild type and P2Y2 knock out osteoblasts. Our findings suggest that P2Y2 receptors play important roles in bone marrow cell differentiation and mineralization as well as in bone cell mechanotransduction, leading to an osteopenic phenotype in P2Y2 knockout mice.

P2Y(2) receptors and GRK2 are involved in oscillatory fluid flow induced ERK1/2 responses in chondrocytes.

Mechanical loading is an important factor regulating cartilage metabolism maintained by chondrocytes. However, some of its underlying mechanisms remain poorly understood. In this study, we employed a chondrogenic cell line ATDC5 to investigate roles of P2Y(2) and GRK2 in chondrocyte mechanotransduction. We first confirmed the expression of chondrocyte markers in differentiated ATDC5 cells. We then exposed both differentiated and undifferentiated ATDC5 cells to oscillatory fluid flow, and found that differentiated ATDC5 cells responded to oscillatory fluid flow by increasing COX-2 and aggrecan expressions. More importantly, fluid flow induced ERK1/2 response in differentiated cells was increased more than 10 times compared to those in undifferentiated cells. Furthermore, we found that P2Y(2) mRNA and protein levels in differentiated ATDC5 cells were significantly higher than those in undifferentiated cells. In contrast, GRK2 protein levels in differentiated cells were significantly lower than those in undifferentiated cells. Finally, overexpressions of P2Y(2) and GRK2 in differentiated ATDC5 cells result in a 34% increase and a 21% decrease of the ERK1/2 phosphorylation, respectively, in response to oscillatory fluid flow, suggesting important roles of P2Y(2) and GRK2 in chondrocyte mechanotransduction.