Faculty Bibliography

Purpose: To assess the use of diffusion-tensor (DT) imaging of articular cartilage to detect and grade early cartilage damage in human specimens with early signs of cartilage damage. Materials and Methods: This study was approved by the institutional review board. Forty-three cartilage-on-bone samples drilled from 21 human patellae were examined with 17.6-T magnetic resonance (MR) imaging and a diffusion-weighted spin-echo sequence (spatial resolution, 50 x 100 x 800 mum). Subsequently, samples underwent histologic analysis with safranin O staining. Cartilage damage on safranin O histologic slides was quantified with Osteoarthritis Research Society International (OARSI) grades; grades ranged from 0 (healthy) to 6 (bone remodeling). Maps of longitudinal diffusivity (lambda(l)), transverse diffusivity (lambda(t)), mean diffusivity (MD), and fractional anisotropy (FA) were calculated. Cartilage was segmented, and region of interest (ROI) analysis was performed and compared with histologic findings. Significant differences in MR parameters between the OARSI groups were assessed with the Tukey test. The value of DT imaging in the diagnosis and grading of cartilage damage was assessed with logistic regression analysis. Results: Samples had OARSI grades of 0 (n = 14), 1 (n = 11), 2 (n = 12), 3 (n = 4), and 4 (n = 2). Samples with an OARSI grade greater than 0 had significantly increased lambda(l), lambda(t), and MD (7%-25% increase) in the superficial cartilage growing deeper into cartilage with increasing OARSI grade. Samples with an OARSI grade greater than 0 showed significantly decreased FA in the deep cartilage (-25% to -35% decrease), suggesting that changes in the collagen architecture may occur early in cartilage degradation. DTI showed excellent performance in the detection of cartilage damage (accuracy, 0.95; 41 of 43 samples) and good performance in the grading of cartilage damage (accuracy, 0.74; 32 of 43 samples). Conclusion: DT imaging of articular cartilage can enable physicians to detect and grade early cartilage damage. (c) RSNA, 2012.

Annexin A6 (AnxA6) is highly expressed in hypertrophic and terminally differentiated growth plate chondrocytes. Rib chondrocytes isolated from newborn AnxA6-/- mice showed delayed terminal differentiation as indicated by reduced terminal differentiation markers, including alkaline phosphatase, matrix metalloproteases-13, osteocalcin, and runx2, and reduced mineralization. Lack of AnxA6 in chondrocytes led to a decreased intracellular Ca(2+) concentration and protein kinase C alpha (PKCalpha) activity, ultimately resulting in reduced extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) activities. The 45 C-terminal amino acids of AnxA6 (AnxA6(1-627)) were responsible for the direct binding of AnxA6 to PKCalpha. Consequently, transfection of AnxA6-/- chondrocytes with full-length AnxA6 rescued the reduced expression of terminal differentiation markers, whereas transfection of AnxA6-/- chondrocytes with AnxA6(1-627) did not or only partially rescued the decreased mRNA levels of terminal differentiation markers. In addition, lack of AnxA6 in matrix vesicles, which initiate the mineralization process in growth plate cartilage, resulted in reduced alkaline phosphatase activity and Ca(2+) and inorganic phosphate (P(i)) content and the inability to form hydroxyapatite-like crystals in vitro. Histological analysis of femoral, tibial, and rib growth plates from newborn mice revealed that the hypertrophic zone of growth plates from newborn AnxA6-/- mice was reduced in size. In addition, reduced mineralization was evident in the hypertrophic zone of AnxA6-/- growth plate cartilage, although apoptosis was not altered compared with wild type growth plates. In conclusion, AnxA6 via its stimulatory actions on PKCalpha and its role in mediating Ca(2+) flux across membranes regulates terminal differentiation and mineralization events of chondrocytes.

Biomineralization is a multifactorial and complex process, which results in the deposition of mineral crystals in the extracellular matrix of various tissues. Physiological mineralization is restricted to tissues, such as bones, teeth, and certain areas of cartilage. Pathological or ectopic mineralization can occur in many soft tissues, including articular cartilage, cardiovascular tissues, kidney, ligaments, and tendons, and can lead to serious problems. Therefore, the understanding of factors and mechanisms that regulate the mineralization process is essential for the development of novel therapeutic strategies to prevent or inhibit ectopic mineralization. This review will discuss some of the mechanisms and factors that regulate physiological mineralization and their potential roles in ectopic mineralization. Finally, potential therapeutic approaches for the treatment of ectopic mineralization are being discussed.

Stem cell application is a burgeoning field of medicine that is likely to influence the future of orthopaedic surgery. Stem cells are associated with great promise and great controversy. For the orthopaedic surgeon, stem cells may change the way that orthopaedic surgery is practiced and the overall approach of the treatment of musculoskeletal disease. Stem cells may change the field of orthopaedics from a field dominated by surgical replacements and reconstructions to a field of regeneration and prevention. This review will introduce the basic concepts of stem cells pertinent to the orthopaedic surgeon and proceed with a more in depth discussion of current developments in the study of stem cells in fracture healing

Extracellular inorganic phosphate (P(i)) concentrations are the highest in the growth plate just before the onset of mineralization. The study reported here demonstrates that P(i) not only is required for hydroxyapatite mineral formation but also modulates terminal differentiation and apoptosis of growth plate chondrocytes. Extracellular P(i) stimulated terminal differentiation marker gene expression, including the progressive ankylosis gene (ank), alkaline phosphatase (APase), matrix metalloproteinase-13 (MMP-13), osteocalcin, and runx2, mineralization, and apoptosis of growth plate chondrocytes. The stimulatory effect of extracellular P(i) on terminal differentiation and apoptosis events of growth plate chondrocytes was dependent on the concentration, the expression levels of type III Na(+)/P(i) cotransporters, and ultimately P(i) uptake. A high extracellular P(i) concentration was required for the stimulation of apoptosis, whereas lower P(i) concentrations were required for the most effective stimulation of terminal differentiation events, including terminal differentiation marker gene expression and mineralization. Suppression of Pit-1 was sufficient to inhibit the stimulatory effects of extracellular P(i) on terminal differentiation events. On the other hand, increasing the local extracellular P(i) concentration by overexpressing ANK, a protein transporting intracellular PP(i) to the extracellular milieu where it is hydrolyzed to P(i) in the presence of APase, resulted in marked increases of hypertrophic and early terminal differentiation marker mRNA levels, including APase, runx2 and type X collagen, and slight increase of MMP-13 mRNA levels, but decreased osteocalcin mRNA level, a late terminal differentiation markers. In the presence of levamisole, a specific APase inhibitor to prevent hydrolysis of extracellular PP(i) to P(i), ANK overexpression of growth plate chondrocytes resulted in decreased mRNA levels of hypertrophic and terminal differentiation markers but increased MMP-13 mRNA levels. In conclusion, with extracellular PP(i) inhibiting and extracellular P(i) stimulating hypertrophic and terminal differentiation events, a precise regulation of PP(i)/P(i) homeostasis is required for the spatial and temporal control of terminal differentiation events of growth plate chondrocytes

OBJECTIVE:: Bone morphogenetic proteins (BMPs) play important roles in the stimulation of osteogenesis and osteoinduction during bone fracture healing and their expression levels may be important for bone graft efficacy. The objective of this study was to determine if there are variations in the expression of BMPs and their receptors in various bone graft harvesting sites. We analyzed autogenous marrow aspirates obtained from three different graft sites for the mRNA levels of BMPs and their receptors. METHODS:: Using real-time polymerase chain reaction, we analyzed the mRNA levels of BMPs and their receptors in autogenous bone marrow aspirates obtained from three different bone graft sites of 10 different human subjects. Collection of autogenous bone marrow from the iliac crest, the proximal humerus, and the proximal tibia was performed using standard sterile techniques in the operating room as part of surgery to treat an established fracture nonunion. RESULTS:: The mRNA levels of BMP-2 and BMP-5 were the highest in the bone marrow aspirates from the three different sites, whereas the mRNA levels of the other osteoinductive BMPs (BMP-4, -5, -6, -7, -8, and -9) were lower. The mRNA levels of BMP-3, an inhibitor of osteogenesis, were the lowest in the bone marrow aspirates of all three different sites. There were no statistical significant differences in the mRNA levels of any of the BMPs or their receptors investigated in this study in the bone marrow of the three different sites. CONCLUSION:: Because no statistical significant differences in the mRNA levels of the BMPs and their receptors were detected in the bone marrow aspirates from the three different sites, our findings suggest that potential differences of various graft sites in the augmentation of bone healing does not result from different expression levels of BMPs

The progressive ankylosis gene (ank) encodes a transmembrane protein that transports intracellular inorganic pyrophosphate (PP(i)) to the extracellular milieu. ank/ank mice, which express a truncated nonfunctional ANK, showed a markedly reduced bone mass, bone-formation rate, and number of tartrate-resistant acid phosphatase-positive (TRAP(+)) multinucleated osteoclasts. ANK function deficiency suppressed osteoblastic differentiation of ank/ank bone marrow stromal cells, as indicated by the decrease in the expression of bone marker genes, including osterix, reduced alkaline phosphatase activity, and mineralization. Runx2 gene expression levels were not altered. Conversely, overexpression of ANK in the preosteoblastic cell line MC3T3-E1 resulted in increased expression of bone marker genes, including osterix. Whereas runx2 expression was not altered in ANK-overexpressing MC3T3-E1 cells, runx2 transcriptional activity was increased. Extracellular PP(i) or P(i) stimulated osteoblastogenic differentiation of MC3T3-E1 cells or partially rescued delayed osteoblastogenic differentiation of ank/ank bone marrow stromal cells. A loss of PP(i) transport function ANK mutation also stimulated osteoblastogenic differentiation of MC3T3-E1 cells. Furthermore, ANK function deficiency suppressed the formation of multinucleated osteoclasts from ank/ank bone marrow cells cultured in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor-kappaB ligand. In conclusion, ANK is a positive regulator of osteoblastic and osteoclastic differentiation events toward a mature osteoblastic and osteoclastic phenotype. (c) 2010 American Society for Bone and Mineral Research

The progressive ankylosis gene (ank) is a transmembrane protein that transports intracellular pyrophosphate to the extracellular milieu. Human mutations of ank lead to craniometaphyseal dysplasia, a disease which is characterized by the overgrowth of craniofacial bones and osteopenia in long bones, suggesting that ANK plays a regulatory role in osteoblast differentiation. To determine the role of ANK in osteoblast differentiation, we suppressed ANK expression in the osteoblastic MC3T3-E1 cell line using siRNA and determined the expression of osteoblastic marker genes and the transcription factors osterix and runx2. In addition, we determined the osteoblastic differentiation of bone marrow stromal cells isolated from the bone marrow of ank/ank mice, which express a truncated, nonfunctional ANK protein, or wild-type littermates. Suppression of ANK expression in MC3T3-E1 cells led to a decrease in bone marker gene expression, including alkaline phosphatase, bone sialoprotein, osteocalcin and type I collagen. In addition, osterix gene expression was decreased in ANK expression-suppressed MC3T3 cells, whereas runx2 expression was increased. Bone marrow stromal cells isolated from ank/ank mice cultured in the presence of ascorbate-2-phosphate for up to 35 days showed markedly reduced mineralization compared to the mineralization of bone marrow stromal cells isolated from wild-type littermates. In conclusion, these findings suggest that ANK is a positive regulator of differentiation events towards a mature osteoblastic phenotype

Physiological mineralization in growth plate cartilage is highly regulated and restricted to terminally differentiated chondrocytes. Since mineralization occurs in the extracellular matrix, we asked whether major extracellular matrix components (collagens) of growth plate cartilage are directly involved in regulating the mineralization process. Our findings show that types II and X collagen interacted with cell surface expressed annexin V. These interactions led to a stimulation of annexin V-mediated Ca2+ influx resulting in an increased intracellular Ca2+ concentration, [Ca2+]i, and ultimately increased alkaline phosphatase activity and mineralization of growth plate chondrocytes. Consequently, stimulation of these interactions (ascorbate to stimulate collagen synthesis, culturing cells on type II collagen-coated dishes, or overexpression of full-length annexin V) resulted in increase of [Ca2+]i, alkaline phosphatase activity and mineralization of growth plate chondrocytes, whereas inhibition of these interactions (3,4-dehydro-L-proline to inhibit collagen secretion, K-201, a specific annexin channel blocker, overexpression of N-terminus deleted mutant annexin V that does not bind to type II collagen and shows reduced Ca2+ channel activities) decreased [Ca2+]i, alkaline phosphatase activity and mineralization. In conclusion, the interactions between collagen and annexin V regulate mineralization of growth plate cartilage. Since annexin V is upregulated during pathological mineralization events of articular cartilage, it is possible that these interactions also regulate pathological mineralization