Individual and cumulative measures of knee joint load associate with T2 relaxation times of knee cartilage in young, uninjured individuals: A pilot study.

BACKGROUND: Articular cartilage structure and chondrocyte health are sensitive and reliant on dynamic joint loading during activities. The purpose of this pilot study was to determine the association between measures of individual and cumulative knee joint loading with T2 relaxation times in the knee cartilage of young individuals without knee injury. METHODS: Twelve participants (17-30 years old) without history of knee injury or surgery completed MRI, physical activity (PA), and biomechanical gait testing. T2 relaxation times were calculated in the cartilage within the patella and lateral and medial compartments. Accelerometry was used to measure mean daily step counts, minutes of PA, and % sedentary time over 7 days. Vertical ground reaction force, external knee joint moments and peak knee flexion angle were measured during stance phase of gait using three-dimensional motion capture. Cumulative knee joint loading was calculated as daily step count by external knee joint moment impulse. The relationship between measures of knee joint loading and T2 relaxation times was assessed using Pearson correlations. RESULTS: Higher T2 relaxation times in the femoral and tibial cartilage were consistently correlated to greater body mass, daily step counts, moderate and vigorous PA, and peak knee joint moments (r = 0.10-0.84). Greater cumulative knee flexion and adduction loading was associated with higher T2 relaxation times in the femoral and tibial cartilage (r = 0.16-0.65). CONCLUSION: Preliminary findings suggest that individual loading factors and cumulative knee joint loading are associated with higher T2 relaxation times in the articular cartilage of young, healthy knees.

Mechanical stimulation of growth plate chondrocytes: Previous approaches and future directions.

Growth plate cartilage resides near the ends of long bones and is the primary driver of skeletal growth. During growth, both intrinsically and extrinsically generated mechanical stresses act on chondrocytes in the growth plate. Although the role of mechanical stresses in promoting tissue growth and homeostasis has been strongly demonstrated in articular cartilage of the major skeletal joints, effects of stresses on growth plate cartilage and bone growth are not as well established. Here, we review the literature on mechanobiology in growth plate cartilage at macroscopic and microscopic scales, with particular emphasis on comparison of results obtained using different methodological approaches, as well as from whole animal and in vitro experiments. To answer these questions, macroscopic mechanical stimulators have been developed and applied to study mechanobiology of growth plate cartilage and chondrocytes. However, the previous approaches have tested a limited number of stress conditions, and the mechanobiology of a single chondrocyte has not been well studied due to limitations of the macroscopic mechanical stimulators. We explore how microfluidics devices can overcome these limitations and improve current understanding of growth plate chondrocyte mechanobiology. In particular, microfluidic devices can generate multiple stress conditions in a single platform and enable real-time monitoring of metabolism and cellular behavior using optical microscopy. Systematic characterization of the chondrocytes using microfluidics will enhance our understanding of how to use mechanical stresses to control the bone growth and the properties of tissue-engineered growth plate cartilage.

Constructive antagonism in limb development.

Several advances have been made in our understanding of the control of the growth and patterning of embryonic limbs. Development of the vertebrate limb is dependent on reciprocal interactions between the ectoderm and mesoderm that regulate the structure and function of the apical ectodermal ridge. One key component of this regulatory program appears to be the precise control of signaling by members of the bone morphogenetic protein family via multiple antagonistic interactions.

Role of BMP family members during kidney development.

Members of the Bone morphogenetic protein (BMP) family have been shown to be important signaling molecules throughout mouse development. Accordingly, many BMPs are also expressed during organogenesis of the metanephric kidney. However, only BMP7 has been shown to be absolutely required for proper formation of the kidney, thus the majority of information known involves this family member. BMP7 is expressed in both the ureteric epithelium and the mesenchyme throughout embryonic development and has been shown to function as a survival factor for the nephrogenic mesenchyme. However, there has been some controversy over the role of BMP7 as an inducing molecule for the metanephric mesenchyme. Recent studies have shown that BMP7 functions as an anti-differentiation factor for this mesenchyme cell population. The function of BMPs in the ureter and in the more differentiated epithelial structures of the nephron is less well understood.

Interaction between FGF and BMP signaling pathways regulates development of metanephric mesenchyme.

Nephrogenesis in the mouse kidney begins at embryonic day 11 and ends approximately 10 days postpartum. During this period, new nephrons are continually being generated from a stem-cell population-the nephrogenic mesenchyme-in response to signals emanating from the tips of the branching ureter. Relatively little is known about the mechanism by which the nephrogenic mesenchyme cell population is maintained at the tips of the ureter in the presence of signals promoting tubulogenesis. Previous studies have shown that a loss of Bmp7 function leads to kidney defects that are a likely result of progressive loss of nephrogenic mesenchyme by apoptosis. The studies presented here demonstrate that BMP7 signaling can prevent apoptosis in explants of metanephric mesenchyme. The surviving mesenchyme cell population, however, is not competent to respond to signals promoting tubulogenesis. In conjunction with FGF2, BMP7 promotes growth and maintains competence of the mesenchyme in vitro. In addition, FGF2 and BMP7 signaling, both independently and in combination, inhibit tubulogenesis. Interestingly, FGF2 and BMP7 also promote expansion of the stromal progenitor cell population in whole kidney culture. Because BMP7 is not produced by stromal progenitor cells, these data suggest a novel interaction between the nephrogenic mesenchyme and stromal progenitor cell populations. A model for the regulation of nephrogenesis by FGF and BMP signaling is presented.

BMP7 acts in murine lens placode development.

Targeted inactivation of the Bmp7 gene in mouse leads to eye defects with late onset and variable penetrance (A. T. Dudley et al., 1995, Genes Dev. 9, 2795-2807; G. Luo et al., 1995, Genes Dev. 9, 2808-2820). Here we report that the expressivity of the Bmp7 mutant phenotype markedly increases in a C3H/He genetic background and that the phenotype implicates Bmp7 in the early stages of lens development. Immunolocalization experiments show that BMP7 protein is present in the head ectoderm at the time of lens placode induction. Using an in vitro culture system, we demonstrate that addition of BMP7 antagonists during the period of lens placode induction inhibits lens formation, indicating a role for BMP7 in lens placode development. Next, to integrate Bmp7 into a developmental pathway controlling formation of the lens placode, we examined the expression of several early lens placode-specific markers in Bmp7 mutant embryos. In these embryos, Pax6 head ectoderm expression is lost just prior to the time when the lens placode should appear, while in Pax6-deficient (Sey/Sey) embryos, Bmp7 expression is maintained. These results could suggest a simple linear pathway in placode induction in which Bmp7 functions upstream of Pax6 and regulates lens placode induction. At odds with this interpretation, however, is the finding that expression of secreted Frizzled Related Protein-2 (sFRP-2), a component of the Wnt signaling pathway which is expressed in prospective lens placode, is absent in Sey/Sey embryos but initially present in Bmp7 mutants. This suggests a different model in which Bmp7 function is required to maintain Pax6 expression after induction, during a preplacodal stage of lens development. We conclude that Bmp7 is a critical component of the genetic mechanism(s) controlling lens placode formation.

Regulation of BMP7 expression during kidney development.

Members of the Bone Morphogenetic Protein (BMP) family exhibit overlapping and dynamic expression patterns throughout embryogenesis. However, little is known about the upstream regulators of these important signaling molecules. There is some evidence that BMP signaling may be autoregulative as demonstrated for BMP4 during tooth development. Analysis of BMP7 expression during kidney development, in conjunction with studies analyzing the effect of recombinant BMP7 on isolated kidney mesenchyme, suggest that a similar mechanism may operate for BMP7. We have generated a beta-gal-expressing reporter allele at the BMP7 locus to closely monitor expression of BMP7 during embryonic kidney development. In contrast to other studies, our analysis of BMP7/lacZ homozygous mutant embryos, shows that BMP7 expression is not subject to autoregulation in any tissue. In addition, we have used this reporter allele to analyze the expression of BMP7 in response to several known survival factors (EGF, bFGF) and inducers of metanephric mesenchyme, including the ureteric bud, spinal cord and LiCl. These studies show that treatment of isolated mesenchyme with EGF or bFGF allows survival of the mesenchyme but neither factor is sufficient to maintain BMP7 expression in this population of cells. Rather, BMP7 expression in the mesenchyme is contingent on an inductive signal. Thus, the reporter allele provides a convenient marker for the induced mesenchyme. Interestingly LiCl has been shown to activate the Wnt signaling pathway, suggesting that BMP7 expression in the mesenchyme is regulated by a Wnt signal. Treatment of whole kidneys with sodium chlorate to disrupt proteoglycan synthesis results in the loss of BMP7 expression in the mesenchyme whereas expression in the epithelial components of the kidney are unaffected. Heterologous recombinations of ureteric bud with either limb or lung mesenchyme demonstrate that expression of BMP7 is maintained in this epithelial structure. Taken together, these data indicate that BMP7 expression in the epithelial components of the kidney is not dependent on cell-cell or cell-ECM interactions with the metanephric mesenchyme. By contrast, BMP7 expression in the metanephric mesenchyme is dependent on proteoglycans and possibly Wnt signaling.

Mice lacking Bmp6 function.

Bmp6, a member of the 60A subgroup of bone morphogenetic proteins (BMPs), is expressed in diverse sites in the developing mouse embryo from preimplantation stages onwards. To evaluate roles for Bmp6 signaling in vivo, gene targeting was used to generate a null mutation at the Bmp6 locus. The resulting Bmp6 mutant mice are viable and fertile, and show no overt defects in tissues known to express Bmp6 mRNA. The skeletal elements of newborn and adult mutants are indistinguishable from wild-type. However, careful examination of skeletogenesis in late gestation embryos reveals a consistent delay in ossification strictly confined to the developing sternum. In situ hybridization studies in the developing long bones and sternum show that other BMP family members are expressed in overlapping domains. In particular we find that Bmp2 and Bmp6 are coexpressed in hypertrophic cartilage, suggesting that Bmp2 may functionally compensate in Bmp6 null mice. The defects in sternum development in Bmp6 null mice are likely to be associated with a transient early expression of Bmp6 in the sternal bands, prior to ossification. These sternal defects are slightly exacerbated in Bmp5/6 double mutant animals.

Overlapping expression domains of bone morphogenetic protein family members potentially account for limited tissue defects in BMP7 deficient embryos.

BMP7 is expressed at diverse sites in the developing mouse embryo, including visceral endoderm, notochord, heart, eye, kidney, and bone. A null mutation in BMP7 results in defects largely confined to the developing kidney and eye. To examine whether other bone morphogenetic protein (BMP) family members potentially substitute for BMP7 in mutant embryos, thereby restricting the observed defects, we analyzed the expression patterns of BMP2 through BMP7 in wild-type and mutant tissues. In the central nervous system and heart, which develop normally in the absence of BMP7 signaling, expression domains of other BMP family members completely overlap with that of BMP7. The variable expressivity of the eye defect correlates with partially overlapping BMP4 and BMP7 expression domains during early eye induction. The loss of BMP7 signaling in the kidney results in apoptosis in the metanephric mesenchyme, a cell population that exclusively expresses BMP7. Thus, tissue defects observed in BMP7 deficient embryos are restricted to cell populations exclusively expressing BMP7. These data suggest that BMP family members can functionally substitute for BMP7 at sites where they colocalize in vivo.

A requirement for bone morphogenetic protein-7 during development of the mammalian kidney and eye.

BMP-7/OP-1, a member of the transforming growth factor-beta (TGF-beta) family of secreted growth factors, is expressed during mouse embryogenesis in a pattern suggesting potential roles in a variety of inductive tissue interactions. The present study demonstrates that mice lacking BMP-7 display severe defects confined to the developing kidney and eye. Surprisingly, the early inductive tissue interactions responsible for establishing both organs appear largely unaffected. However, the absence of BMP-7 disrupts the subsequent cellular interactions required for their continued growth and development. Consequently, homozygous mutant animals exhibit renal dysplasia and anophthalmia at birth. Overall, these findings identify BMP-7 as an essential signaling molecule during mammalian kidney and eye development.