Xuanhao Sun

Tenogenic differentiation of human MSCs induced by the topography of electrochemically aligned collagen threads

Topographical cues from the extracellular microenvironment can influence cellular activity including proliferation and differentiation. Information on the effects of material topography on tenogenic differentiation of human mesenchymal stem cells (human MSCs) is limited. A methodology using the principles of isoelectric focusing has previously been developed in our laboratory to synthesize electrochemically aligned collagen (ELAC) threads that mimics the packing density, alignment and strength of collagen dense connective tissues. In the current study, human MSCs were cultured on ELAC and randomly oriented collagen threads and the effect of collagen orientation on cell morphology, proliferation and tenogenic differentiation was investigated. The results indicate that higher rates of proliferation were observed on randomly oriented collagen threads compared to ELAC threads. On the other hand, tendon specific markers such as scleraxis and tenomodulin, were significantly increased on ELAC threads compared to randomly oriented collagen threads. Additionally, osteocalcin, a specific marker of bone differentiation was suppressed on ELAC threads. Previous studies have reported that BMP-12 is a key growth factor to induce tenogenic differentiation of MSCs. To evaluate the synergistic effect of BMP-12 and collagen orientation, human MSCs were cultured on ELAC threads in culture medium supplemented with and without BMP-12. The results revealed that BMP-12 did not have an additional effect on the tenogenic differentiation of human MSCs on ELAC threads. Together, these results suggest that ELAC induces tenogenic differentiation of human MSCs by presenting an aligned and dense collagen substrate, akin to the tendon itself. In conclusion, ELAC has a significant potential to be used as a tendon replacement and in the development of an osteotendinous construct towards the regeneration of bone-tendon interfaces.

Random lasing in bone tissue

Owing to the low-loss and high refractive index variations derived from the basic building block of bone structure, we, for the first time to our knowledge, demonstrate coherent random lasing action originated from the bone structure infiltrated with laser dye, revealing that bone tissue is an ideal biological material for random lasing. Our numerical simulation shows that random lasers are extremely sensitive to subtle structural changes even at nanoscales and can potentially be an excellent tool for probing nanoscale structural alterations in real time as a novel spectroscopic modality.

Visualization of a phantom post-yield deformation process in cortical bone

A prominent opacity is evident in the process zone of notched thin wafers of bone loaded in tension. Being recoverable upon unloading, this opaque zone can be stained only when the sample is under load, unlike the classically reported forms of damage which take up the stain in the unloaded state. Furthermore, despite the stain uptake, microcracks are absent in the stained area examined by high magnification optical microscopy and atomic force microscopy (AFM). Therefore, the size scale and the electric charge of the features involved in the process zone were probed at the submicron level by using a wide range of fluorescent dyes of different molecular weights and charges. It was observed that negatively charged dyes penetrate the process zone and that dyes greater than 10 kDa (about 10-20 nm in size) were unable to label the process zone. Digital image correlation (DIC) measurements indicated that the opacity initiates at about 1% principal strain and the strain accumulates up to 14%. While the opacity was largely recoverable upon unloading, the core regions which experienced large strains had permanent residual strains up to 2%, indicating that the observed deformation phenomenon can be interlocked within bone matrix without the formation of microcracks. Based on the similarity of size and their known affinity for negatively charged species, exposure of mineral nanoplatelets is proposed as prime candidates. Therefore, the deformation process reported here may be associated with debonding of mineral crystals from the neighboring collagen molecules. Overall, post-yield deformation of bone at the micron scale takes place by large strain events which are accommodated in bone matrix by the generation of nanoscale positively charged interfaces.

Detection of nanoscale structural changes in bone using random lasers

We demonstrate that the unique characteristics of random lasing in bone can be used to assess nanoscale structural alterations as a mechanical or structural biosensor, given that bone is a partially disordered biological nanostructure. In this proof-of-concept study, we conduct photoluminescence experiments on cortical bone specimens that are loaded in tension under mechanical testing. The ultra-high sensitivity, the large detection area, and the simple detection scheme of random lasers allow us to detect prefailure damage in bone at very small strains before any microscale damage occurs. Random laser-based biosensors could potentially open a new possibility for highly sensitive detection of nanoscale structural and mechanical alterations prior to overt microscale changes in hard tissue and biomaterials.

Mechanical Stretch Induced Calcium Efflux from Bone Matrix Stimulates Osteoblasts

The mechanisms by which bone cells sense critically loaded regions of bone are still a matter of ongoing debate. Animal models to investigate response to microdamage involve post mortem immunohistological analysis and do not allow real-time monitoring of cellular response during the emergence of the damage in bone. Most in vitro mechanical stimulation studies are conducted on non-bone substrates, neglecting the damage-related alterations in the pericellular niche and their potential effects on bone cells. The current study reports spontaneous efflux of calcium ions (Ca(2+)) (1.924±0.742 pmol cm(-2)s(-1)) from regions of devitalized bone matrix undergoing post-yield strains, induced by a stress concentrator. When these samples are seeded with MC3T3-E1 osteoblasts, the strain-induced Ca(2+) efflux from bone elicits cell response at the stress concentration site as manifested by activation of intracellular calcium signaling (increase in fluorescence by 52%±27%). This activity is associated with extracellular calcium because the intracellular calcium signaling in response to mechanical loading subsides when experiments are repeated using demineralized bone substrates (increase in fluorescence by 6%±10%). These results imply a novel perspective where bone matrix acts as an intermediary mechanochemical transducer by converting mechanical strain into a chemical signal (pericellular calcium) to which cells respond. Such a mechanism may be responsible for triggering repair at locations of bone matrix undergoing critical deformation levels.

Spectroscopic visualization of nanoscale deformation in bone: interaction of light with partially disordered nanostructure

Given that bone is an intriguing nanostructured dielectric as a partially disordered complex structure, we apply an elastic light scattering-based approach to image prefailure deformation and damage of bovine cortical bone under mechanical testing. We demonstrate that our imaging method can capture nanoscale deformation in a relatively large area. The unique structure, the high anisotropic property of bone, and the system configuration further allow us to use the transfer matrix method to study possible spectroscopic manifestations of prefailure deformation. Our sensitive yet simple imaging method could potentially be used to detect nanoscale structural and mechanical alterations of hard tissue and biomaterials in a fairly large field of view.

Novel mechanical bioreactor for concomitant fluid shear stress and substrate strain

The two main types of mechanical stimuli used in cellular-level bone mechanotransduction studies are substrate strain and flow-induced shear stress. A subset of studies has investigated which of these stimuli induces the primary mechanotransduction effect on bone cells. The shortcomings of these experiments are twofold. First, in some experiments the magnitude of one loading type is able to be quantitatively measured while the other loading mode is only estimated. Second, the two loading modes are compared using different bioreactors, representing different cellular environments and substrates to which the cells are attached. In addition, none of these studies utilized bioreactors which apply controlled magnitudes of substrate strain and flow-induced shear stress differentially and simultaneously. This study presents the design of a multimodal loading device which can apply substrate stretch and fluid flow simultaneously while allowing for real-time cell imaging. The mechanical performance of the bioreactor is validated in this study by correlating the output levels of flow-induced shear stress and substrate strain with the input levels of displacement and displacement rate. The magnitudes of cross-talk loading (i.e. flow-induced strain, and strain-induced fluid flow) are also characterized and shown to be magnitudes lower than physiological levels of loading estimated to occur in bone in vivo.

Osteoblasts detect pericellular calcium concentration increase via neomycin-sensitive voltage gated calcium channels

The mechanisms underlying the detection of critically loaded or micro-damaged regions of bone by bone cells are still a matter of debate. Our previous studies showed that calcium efflux originates from pre-failure regions of bone matrix and MC3T3-E1 osteoblasts respond to such efflux by an increase in the intracellular calcium concentration. The mechanisms by which the intracellular calcium concentration increases in response to an increase in the pericellular calcium concentration are unknown. Elevation of the intracellular calcium may occur via release from the internal calcium stores of the cell and/or via the membrane bound channels. The current study applied a wide range of pharmaceutical inhibitors to identify the calcium entry pathways involved in the process: internal calcium release from endoplasmic reticulum (ER, inhibited by thapsigargin and TMB-8), calcium receptor (CaSR, inhibited by calhex), stretch-activated calcium channel (SACC, inhibited by gadolinium), voltage-gated calcium channels (VGCC, inhibited by nifedipine, verapamil, neomycin, and ω-conotoxin), and calcium-induced-calcium-release channel (CICRC, inhibited by ryanodine and dantrolene). These inhibitors were screened for their effectiveness to block intracellular calcium increase by using a concentration gradient induced calcium efflux model which mimics calcium diffusion from the basal aspect of cells. The inhibitor(s) which reduced the intracellular calcium response was further tested on osteoblasts seeded on mechanically loaded notched cortical bone wafers undergoing damage. The results showed that only neomycin reduced the intracellular calcium response in osteoblasts, by 27%, upon extracellular calcium stimulus induced by concentration gradient. The inhibitory effect of neomycin was more pronounced (75% reduction in maximum fluorescence) for osteoblasts seeded on notched cortical bone wafers loaded mechanically to damaging load levels. These results imply that the increase in intracellular calcium occurs by the entry of extracellular calcium ions through VGCCs which are sensitive to neomycin. N-type and P-type VGCCs are potential candidates because they are observed in osteoblasts and they are sensitive to neomycin. The calcium channels identified in this study provide new insight into mechanisms underlying the targeted repair process which is essential to bone adaptation.

A difference imaging technique for monitoring real-time changes in morphology within the cell, tissue, and organism spatial domain

Image subtraction has been an extremely useful tool for capturing subtle changes in pixel intensity with extremely high temporal resolution, and has been used for decades in the astronomy and metal corrosion fields. However, to date, image subtraction has not been used as a mainstream technique for investigating morphological changes in cells, tissues, or whole organisms. We introduce a user-friendly differential imaging technique for monitoring real time (~msec) changes in morphology within the micrometer to millimeter spatial scale. The technique is demonstrated by measuring morphological changes morphology for biomedical (bone stress), agricultural (crop root elongation), and environmental (zooplankton ecotoxicology) applications. Subtle changes in growth that would typically only be observed by highly skilled experts are easily resolved via image subtraction and the use of convolution kernels. When coupled with techniques characterizing real time biochemical transport (e.g., respiration, ion/substrate transport), physiology can be directly quantified with a high temporal and spatial resolution. Because of the ease of use, this technique can be readily applied to any field of science concerned with bridging the gap between form and function.

Elevated solute transport at sites of diffuse matrix damage in cortical bone: Implications on bone repair

Diffuse matrix damage in rat cortical bone has been observed to self‐repair efficiently in 2 weeks without activating bone remodeling, and unlike the case with linear cracks, the local osteocytes at the sites of diffuse damage remain healthy. However, the reason(s) for such high efficiency of matrix repair remains unclear. We hypothesized that transport of minerals and other compounds essential for damage repair is enhanced at the damaged sites and further increased by the application of tensile loading. To test our hypothesis, diffuse damage was introduced in notched bovine wafers under cyclic tensile loading and unloading. Using the Fluorescence Recovery After Photobleaching (FRAP) approach, we measured the transport of a small fluorescent tracer (sodium fluorescein, 376 Da) in damaged versus undamaged regions and under varying tensile load magnitudes (0.2 N, 10 N, 20 N, and 30 N), which corresponded to nominal strains of 12.5, 625, 1,250, and 1,875 microstrains, respectively. We found a 37% increase in transport of fluorescein in damaged regions relative to undamaged regions and a further ∼18% increase in transport under 20 N and 30 N tension compared to the non‐loaded condition, possibly due to the opening of the cracking surfaces. The elevated transport of minerals and other adhesive proteins may, at least partially, account for the highly effective repair of diffuse damage observed in vivo. Clinical Significance: Diffuse damage adversely affects bones fracture resistance and this study provided quantitative data on elevated transport, which may be involved in repairing diffuse damage in vivo. 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:692–698, 2018.