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Biological cell lasers are emerging as a novel technology in biological studies and biomedical engineering. The heterogeneity of cells, however, can result in various lasing behaviors from cell to cell. Thus, the capability to track individual cells during laser investigation is highly desired. In this work, a microwell array was integrated with high-quality Fabry-Pérot cavities for addressable and automated cell laser studies. Cells were captured in the microwells and the corresponding cell lasing was achieved and analyzed using SYTO9-stained Sf9 cells as a model system. It is found that the presence of the microwells does not affect the lasing performance, but the cell lasers exhibit strong heterogeneity due to different cell sizes, cycle stages and polyploidy. Time series laser measurements were also performed automatically with the integrated microarray, which not only enables the tracking and multiplexed detection of individual cells, but also helps identify "abnormal" cells that deviate from a large normal cell population in their lasing performance. The microarrayed cell laser platform developed here could provide a powerful tool in single cell analysis using lasing emission that complements conventional fluorescence-based cell analysis.

Inorganic phosphate (Pi) has been recognized as an important signaling molecule that modulates chondrocyte maturation and cartilage mineralization. However, conclusive experimental evidence for its involvement in early chondrogenesis is still lacking. Here, using high-density monolayer (2D) and pellet (3D) culture models of chondrogenic ATDC5 cells, we demonstrate that the cell response to Pi does not correlate with the Pi concentration in the culture medium but is better predicted by the availability of Pi on a per cell basis (Pi abundance). Both culture models were treated with ITS+, 10mM β-glycerophosphate (βGP), or ITS+/10mM βGP, which resulted in three levels of Pi abundance in cultures: basal (Pi/DNA10ng/µg), moderate (Pi/DNA=25.3 - 32.3ng/µg), and high abundance (Pi/DNA60ng/µg). In chondrogenic medium alone, the abundance levels were at the basal level in 2D culture and moderate in 3D cultures. The addition of 10mM βGP resulted in moderate abundance in 2D and high abundance in 3D cultures. Moderate Pi abundance enhanced early chondrogenesis and production of aggrecan and type II collagen whereas high Pi abundance inhibited chondrogenic differentiation and induced rapid mineralization. Inhibition of sodium phosphate transporters reduced phosphate-induced expression of chondrogenic markers. When 3D ITS+/βGP cultures were treated with levamisole to reduce ALP activity, Pi abundance was decreased to moderate levels, which resulted in significant upregulation of chondrogenic markers, similar to the response in 2D cultures. Delay of phosphate delivery until after early chondrogenesis occurs (7 days) no longer enhanced chondrogenesis, but instead accelerated hypertrophy and mineralization. Together, our data highlights the dependence of chondroprogenitor cell response to Pi on its availability to individual cells and the chondrogenic maturation stage of these cells and suggest that appropriate temporal delivery of phosphate to ATDC5 cells in 3D cultures represents a rapid model for mechanistic studies into the effects of exogenous cues on chondrogenic differentiation, chondrocyte maturation, and matrix mineralization.

Rat tibial growth plates have X-ray opaque tethers that link the epiphysis and metaphysis and increase with age as the growth plate (GP) becomes thinner. To determine if tether formation is a regulated process of GP maturation, we tested the hypotheses that tether properties and distribution can be quantified by micro-computed tomography (microCT), that rachitic GPs typical of vitamin D receptor knockout (VDR(-/-)) mice have fewer tethers and altered tether distribution, and that tether formation is regulated by signaling via the VDR. Distal femoral GPs from VDR(+/+) and VDR(-/-) 8-week-old mice were analyzed with microCT and then processed for decalcified and undecalcified histomorphometry. A wide range of parameters that assessed GP and tether geometry and morphology, along with tether distribution, were measured using both microCT and histology. Growth plates of 10-week-old VDR(+/+) and VDR(-/-) mice on a high-calcium, phosphorus, lactose, and vitamin D(3) rescue diet were also analyzed. Both microCT and histology showed tethers present throughout normal mice GPs, while reduction in tether number and volume percentage occurred in VDR(-/-) GPs with localization to the central region. Decreased shrinkage in the axial direction during decalcified histological processing correlated with tether formation, suggesting mechanical stability due to tethers. Tether formation increased greatly between 8 and 10 weeks. Rescue diets restored VDR(-/-) GP size but not tether volume percentage. Overall, these results demonstrate microCT imaging's utility for analyzing tether formation and suggest that signaling via the VDR plays a pivotal role in tether formation.

The aim of this study was to investigate the effects of alginate and agarose on the response of bone marrow stromal cells (BMSCs) to chondrogenic stimuli. Rat BMSCs were expanded in monolayer culture with or without FGF-2 supplementation. Cells were then seeded in 2% alginate and agarose gels and cultured in media with or without TGF-beta1 or dexamethasone (Dex). Sulfated glycosaminoglycans (sGAGs), collagen type II, and aggrecan were expressed in all groups that received TGF-beta1 treatment during hydrogel culture. Expansion of rat BMSCs in the presence of FGF-2 increased production of sGAG in TGF-beta1-treated groups over those cultures that were treated with TGF-beta1 alone in alginate cultures. However, in agarose, cells exposed to FGF-2 during expansion produced less sGAG within TGF-beta1-supplemented groups over those cultures treated with TGF-beta1 alone. Dex was required for optimal matrix synthesis in both hydrogels, but was found to decrease cell viability in agarose constructs. These results indicate that the response of BMSCs to a chondrogenic growth factor regimen is scaffold dependent.

Improve functional quality of cartilage tissue engineered from stem cells requires a better understanding of the functional evolution of native cartilage tissue. Therefore, a biosynthetic hydrogel was developed containing RGD, hyaluronic acid and/or type-I collagen conjugated to poly(ethylene glycol) acrylate to recapitulate the precondensation microenvironment of the developing limb. Conjugation of any combination of the three ligands did not alter the shear moduli or diffusion properties of the PEG hydrogels; thus, the influence of ligand composition on chondrogenesis could be investigated in the context of varying matrix stiffness. Gene expression of ligand receptors (CD44 and the b1-integrin) as well as markers of condensation (cell clustering and N-cadherin gene expression) and chondrogenesis (Col2a1 gene expression and sGAG production) by chondroprogenitor cells in this system were modulated by both matrix stiffness and ligand composition, with the highest gene expression occurring in softer hydrogels containing all three ligands. Cell proliferation in these 3D matrices for 7 d prior to chondrogenic induction increased the rate of sGAG production in a stiffness-dependent manner. This biosynthetic hydrogel supports the features of early limb-bud condensation and chondrogenesis and is a novel platform in which the influence of the matrix physicochemical properties on these processes can be elucidated.

Skeletogenesis is an exquisitely orchestrated and dynamic process, culminating in the formation of highly variable and complex mineralized structures that are optimized for their function. While cellular and molecular biology studies have provided tremendous recent progress toward understanding how patterns of bone formation are regulated, high resolution imaging techniques such as microcomputed tomography (micro-CT) can provide complementary quantitative information about the progressive changes in three-dimensional (3-D) skeletal morphology and density that occur during early skeletal development and postnatal growth. Furthermore, recently developed in vivo micro-CT systems promise to be a powerful and efficient tool for noninvasively monitoring normal skeletogenesis, as well as for evaluating the effects of genetic or environmental manipulation. This review focuses on the use of micro-CT imaging and analysis to better understand normal and abnormal skeletal development and growth.

The goal of this study was to determine the effects of chondrogenic predifferentiation on the ability of bone marrow-derived stromal cells (BMSCs) delivered to growth plate defects to restore growth function. Chondrogenesis was induced with transforming growth factor (TGF)-β1 treatment in high-density monolayer cultures of BMSCs in vitro. The predifferentiated or undifferentiated BMSCs were either seeded into agarose gels for continued in vitro culture, or injected into growth plate defects via an in situ gelling agarose. Predifferentiated BMSCs had higher Sox-9, type II collagen, and aggrecan mRNA levels compared to undifferentiated cells after high-density monolayer culture. After transfer to agarose gels, predifferentiated cells did not produce a cartilaginous matrix, even with continued TGF-β1 stimulation, whereas undifferentiated cells produced a cartilaginous matrix in this system. Three-dimensional images of the growth plate created from microcomputed tomography scans showed that delivery of either predifferentiated or undifferentiated cells to defects resulted in a decrease in mineralized tether formation (fusion) in the growth plate tissue surrounding the defect to normal levels. Limb length discrepancy between injured and control limbs was corrected after treatment with undifferentiated, but not predifferentiated, cells. These results indicate that cell therapy may be an effective treatment to reduce growth dysfunction after growth plate injury, perhaps by maintaining the health of the uninjured growth plate tissue, and that the cell differentiation state plays a role in restoring the growth potential of the injured limb.

Understanding how the material properties of bone tissue from the various forms of osteogenesis imperfecta (OI) differ will allow us to tailor treatment regimens for affected patients. To this end, we characterized the bone structure and material properties of two mouse models of OI, the osteogenesis imperfecta mouse (oim/oim) and fragilitas ossium (fro/fro), in which bone fragility is due to a genetic defect in collagen type I and a defect in osteoblast matrix mineralization, respectively. Bones from 3 to 6 month old animals were examined using Fourier transform infrared spectroscopic imaging (FTIRI), microcomputed tomography (micro-CT), histology, and biochemical analysis. The attributes of oim/oim bone tissue were relatively constant over time when compared to wild type animals. The mineral density in oim/oim cortices and trabecular bone was higher than wild type while the bones had thinner cortices and fewer trabeculae that were thinner and more widely spaced. The fro/fro animals exhibited osteopenic attributes at 3 months. However, by 6 months, their spectroscopic and geometric properties were similar to wild type animals. Despite the lack of a specific collagen defect in fro/fro mice, both fro/fro and oim/oim genotypes exhibited abnormal collagen crosslinking as determined by FTIRI at both time points. These results demonstrate that abnormal extracellular matrix assembly plays a role in the bone fragility in both of these models.

Osteoporosis is a frequent problem in disorders characterized by iron overload, such as the thalassemias and hereditary hemochromatosis. The exact role of iron in the development of osteoporosis in these disorders is not established. To define the effect of iron excess in bone, we generated an iron-overloaded mouse by injecting iron dextran at 2 doses into C57/BL6 mice for 2 months. Compared with the placebo group, iron-overloaded mice exhibited dose-dependent increased tissue iron content, changes in bone composition, and trabecular and cortical thinning of bone accompanied by increased bone resorption. Iron-overloaded mice had increased reactive oxygen species and elevated serum tumor necrosis factor-α and interleukin-6 concentrations that correlated with severity of iron overload. Treatment of iron-overloaded mice with the antioxidant N-acetyl-L-cysteine prevented the development of trabecular but not cortical bone abnormalities. This is the first study to demonstrate that iron overload in mice results in increased bone resorption and oxidative stress, leading to changes in bone microarchitecture and material properties and thus bone loss.

Injuries to the growth plate remain a significant clinical challenge. The need to better understand mechanisms of growth disruption following transphyseal injuries and evaluate new therapeutic approaches to growth restoration motivates development of a well characterized model of growth plate injury. The goals of this study were to develop a growth plate defect model in the rat and to use microcomputed tomography (micro-CT) imaging to detect and quantify associated changes in growth plate morphology and mineralization over time following injury and in response to treatment. Three-dimensional images of the growth plate were created from micro-CT scans and used to quantify the volume of mineralized tissue within the defect site. Growth plate thickness and volume as well as the degree of growth plate fusion were also measured from the reconstructed 3D images. Growth deficiency was then quantified as a function of time post-injury from whole limb micro-CT scans. Finally, this model was used to determine the ability of an injectable in situ gelling hydrogel to prevent formation of a bony bridge within the defect and the subsequent effect on limb length deficiency and changes to growth plate morphology. Growth plate injury resulted in significant shortening of the defect limb by day 28 and significant thinning and fusion of the surrounding growth plate up to day 112. Limb length reduction was correlated with changes in the growth plate volume and average thickness at day 56. Injection of an in situ gelling agarose into the defect resulted in a reduction of limb length discrepancy as well as a thicker growth plate on average compared to empty defect controls. These results establish a novel method of characterizing changes in whole bone and growth plate morphology due to a growth plate injury and indicate that treatment with agarose hydrogel reduces limb length discrepancy but is not sufficient to regenerate growth plate tissue or fully restore growth function.