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OBJECTIVE: Accelerated osteoclastic bone resorption plays a central role in the pathogenesis of osteoporosis and other bone diseases. Because identifying the molecular pathways that regulate osteoclast activity provides a key to understanding the causes of these diseases and developing new treatments, we studied the effect of adenosine A(1) receptor blockade or deletion on bone density. METHODS: The bone mineral density (BMD) in adenosine A(1) receptor-knockout (A(1)R-knockout) mice was analyzed by dual x-ray absorptiometry (DXA) scanning, and the trabecular and cortical bone volume was determined by microfocal computed tomography (micro-CT). The mice were ovariectomized or sham-operated, and 5 weeks after surgery, when osteopenia had developed, several parameters were analyzed by DXA scanning and micro-CT. A histologic examination of bones obtained from A(1)R-knockout and wild-type mice was carried out. Visualization of osteoblast function (bone formation) after tetracycline double-labeling was performed by fluorescence microscopy. RESULTS: Micro-CT analysis of bones from A(1)R-knockout mice showed significantly increased bone volume. Electron microscopy of bones from A(1)R-knockout mice showed the absence of ruffled borders of osteoclasts and osteoclast bone resorption. Immunohistologic analysis demonstrated that although osteoclasts were present in the A(1)R-knockout mice, they were smaller and often not associated with bone. No morphologic changes in osteoblasts were observed, and bone-labeling studies revealed no change in the bone formation rates in A(1)R-knockout mice. CONCLUSION: These results suggest that the adenosine A(1) receptor may be a useful target in treating diseases characterized by excessive bone turnover, such as osteoporosis and prosthetic joint loosening. [ABSTRACT FROM AUTHOR]

Purpose: The distinction between the dorsal intercarpal (DIC) and dorsal scaphotriquetral (DST) ligaments is imprecise and unclear in the literature. The purpose of our cadaveric study was to define the origins, insertions, and anatomic relationships of the dorsal wrist ligaments and relate these anatomic findings to magnetic resonance imaging (MRI) scans and histology., Methods: The study included 17 unmatched fresh-frozen cadaveric specimens (7 male and 10 female), with a mean age of 67.1 years (range, 48-86 years). Wrists with arthritis or carpal malalignment were excluded. Ligaments were dissected and insertion sites were recorded in the radioulnar (width) and proximodistal (length) dimensions, centered at the midpoints of the insertion. Three cadaveric specimens underwent a histologic analysis to demonstrate ligament composition and insertion sites. Three additional cadavers underwent MRI, from which 3-dimensional models were built to model ligament topography., Results: The conjoined triquetral insertion of the DIC, DST, and dorsal radiocarpal (DRC) measured 88.5 ± 6.4 mm 2 . In each specimen, there were 2 distinct deep and superficial components of intercarpal fibers. The deep component inserted on the lunate with an area of 59.0 ± 5.0 mm 2 . The deep and superficial components diverged as they coursed radially. The superficial component proceeded to the scaphoid ridge, trapezium, and trapezoid, whereas the deep component inserted on the proximal row. The deep fibers blended distally from their lunate insertion with the DST, forming a robust, 2.9 ± 0.8-mm wide extension over the dorsal capitate. The DRC inserted on the lunate, proximal to the DIC and DST insertions, with an area of 23.9 ± 5.4 mm 2 ., Conclusions: The dorsal ligament complex forms a firm link across the proximal carpal row and the DST provides extension of the proximal row over the capitate., Clinical Relevance: This information can guide surgeons while performing a dorsal approach to the wrist and repairing traumatic ligament disruption., (Copyright © 2022 American Society for Surgery of the Hand. Published by Elsevier Inc. All rights reserved.)

Relative to the general population, Native Americans (NA) bear a disproportionate burden of suicide-related mortality rates. NA males and females aged 15 to 24 years experience suicide rates nearly 3 times than the U.S. all races rates in this age group. Although efforts have been made to understand and reduce suicide in tribal communities, a large portion has focused on individual characteristics with less attention given to social factors that may also inform suicide. This article aims to build on a local conceptual model of NA youth suicide by examining additional potential social factors through qualitative interviews. Findings from the thematic analysis resulted in the identification of seven perceived social influences: contagion, violence and abuse, discrimination and bullying, negative expectations, spirituality, social support, and cultural strengths. Public health approaches to reduce suicide should consider local social factors that resonate with tribal communities to build resilience.

Anemia is a frequent complication of chronic kidney disease (CKD), related in part to the disruption of iron metabolism. Iron therapy is very common in children with CKD and excess iron has been shown to induce bone loss in non-CKD settings, but the impact of iron on bone health in CKD remains poorly understood. Here, we evaluated the effect of oral and parenteral iron therapy on bone transcriptome, bone histology and morphometry in two mouse models of juvenile CKD (adenine-induced and 5/6-nephrectomy). Both modalities of iron therapy effectively improved anemia in the mice with CKD, and lowered bone Fgf23 expression. At the same time, iron therapy suppressed genes implicated in bone formation and resulted in the loss of cortical and trabecular bone in the mice with CKD. Bone resorption was activated in untreated CKD, but iron therapy had no additional effect on this. Furthermore, we assessed the relationship between biomarkers of bone turnover and iron status in a cohort of children with CKD. Children treated with iron had lower levels of circulating biomarkers of bone formation (bone-specific alkaline phosphatase and the amino-terminal propeptide of type 1 procollagen), as well as fewer circulating osteoblast precursors, compared to children not treated with iron. These differences were independent of age, sex, and glomerular filtration rate. Thus, iron therapy adversely affected bone health in juvenile mice with CKD and was associated with low levels of bone formation biomarkers in children with CKD., (Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Objectives: Investigate the association of sleep characteristics with suicidal ideation and suicide attempt among middle-aged and older adults with depressive symptoms in five low- and middle-income countries (LMICs)., Design: Cross-sectional., Setting: China, Ghana, India, Russia, and South Africa., Participants: Adults aged ≥50 years with depressive symptoms from the World Health Organization (WHO) Study on Global AGEing and Adult Health (n=2,040)., Measurements: Predictors were self-reported average sleep duration for the past 2 nights (<7 hours (shorter), 7 to <9 hours (reference), ≥9 hours (longer)), sleep quality for the past 2 nights (moderate/good/very good [both nights], poor/very poor [≥1 night]), past-month insomnia symptoms (none/mild, moderate, severe/extreme), and past-day daytime sleepiness. Outcomes were past-year suicidal ideation and suicide attempt. Analyses were adjusted for age, sex, household wealth, marital status, self-rated health, cognitive performance, number of depressive symptoms, and country of residence., Results: Participants with poor/very poor sleep quality ≥1 night had greater odds of suicidal ideation (vs. moderate/good/very good sleep quality both nights). Participants with moderate and severe/extreme insomnia symptoms had greater odds of suicidal ideation and suicide attempt (vs. none/mild insomnia symptoms). In moderation analyses, greater insomnia symptoms were associated with higher odds of suicidal ideation among women only and those aged 60-60 years and ≥80 years only., Conclusions: Among middle-aged and older adults with depressive symptoms in LMICs, sleep characteristics are markers of-and potential contributors to-suicidal ideation and suicide attempt, and there was evidence of moderation by age and sex. Interventions aimed at preventing suicide-related outcomes in these populations should consider the role of sleep., (Copyright © 2019 National Sleep Foundation. All rights reserved.)

Purpose: To compare recovery in a rat model of sciatic nerve injury using a novel polyglycolic acid (PGA) conduit, which contains collagen fibers within the tube, as compared with both a hollow collagen conduit and nerve autograft. We hypothesize that a conduit with a scaffold will provide improved nerve regeneration over hollow conduits and demonstrate no significant differences when compared with autograft., Methods: A total of 72 Sprague-Dawley rats were randomized into 3 experimental groups, in which a unilateral 10-mm sciatic defect was repaired using either nerve autograft, a hollow collagen conduit, or a PGA collagen-filled conduit. Outcomes were measured at 12 and 16 weeks after surgery, and included bilateral tibialis anterior muscle weight, voltage and force maximal contractility, assessment of ankle contracture, and nerve histology., Results: In all groups, outcomes improved between 12 and 16 weeks. On average, the autograft group outperformed both conduit groups, and the hollow conduit demonstrated improved outcomes when compared with the PGA collagen-filled conduit. Differences in contractile force, however, were significant only at 12 weeks (autograft > hollow collagen conduit > PGA collagen-filled conduit). At 16 weeks, contractile force demonstrated no significant difference but corroborated the same absolute results (autograft > hollow collagen conduit > PGA collagen-filled conduit)., Conclusions: Nerve repair using autograft provided superior motor nerve recovery over the 2 conduits for a 10-mm nerve gap in a murine acute transection injury model. The hollow collagen conduit demonstrated superior results when compared with the PGA collagen-filled conduit., Clinical Relevance: The use of a hollow collagen conduit provides superior motor nerve recovery as compared with a PGA collagen-filled conduit., (Copyright © 2019 American Society for Surgery of the Hand. Published by Elsevier Inc. All rights reserved.)

Reduced mechanical loading can lead to disuse osteoporosis, resulting in bone fragility. Disuse models report macroscopic bone loss due to muscle inactivity and immobilization, yet only recently has there been quantification of the effects of disuse on the vascular pores and osteocyte network, which are believed to play an important role in mechanotransduction via interstitial fluid flow. The goal of this study was to perform a high-resolution analysis of the effects of muscle inactivity on intracortical porosity and osteocyte lacunar density in skeletally mature rats. Muscle paralysis was induced in 20-week-old female Sprague Dawley rats by injection of botulinum neurotoxin. Rats were injected in the right hindlimb muscles with either Botox (BTX, n = 8) or saline solution (CTRL, n = 8), with a third group used as baseline controls (n = 8). Four weeks after injection, Botox caused a ∼60% reduction in hindlimb muscle mass. High-resolution micro-CT analysis showed that Botox-induced muscle paralysis increased vascular canal porosity and reduced osteocyte lacunar density within the tibial metaphysis cortex. Cortical thickness and other areal properties were diminished in the proximal tibial metaphysis, whereas no differences were found in the mid-diaphysis. Within the BTX group, the injected limbs showed a lower cancellous bone volume fraction relative to the contralateral limb. These results indicate that diminished muscle activity alters the vascular canal porosity and osteocyte lacunar density in cortical bone, which could alter interstitial fluid flow, affecting molecular transport and the transmission of mechanical signals to osteocytes. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res., (© 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Cartilage injury, such as full-thickness lesions, predisposes patients to the premature development of osteoarthritis, a degenerative joint disease. While surgical management of cartilage lesions has improved, long-term clinical efficacy has stagnated, owing to the lack of hyaline cartilage regeneration and inadequate graft-host integration. This study tests the hypothesis that integration of cartilage grafts with native cartilage can be improved by enhancing the migration of chondrocytes across the graft-host interface via the release of chemotactic factor from a degradable polymeric mesh. To this end, a polylactide-co-glycolide/poly-ε-caprolactone mesh was designed to localize the delivery of insulin-like growth factor 1 (IGF-1), a well-established chondrocyte attractant. The release of IGF-1 (100 ng/mg) enhanced cell migration from cartilage explants, and the mesh served as critical structural support for cell adhesion, growth, and production of a cartilaginous matrix in vitro, which resulted in increased integration strength compared with mesh-free repair. Further, this neocartilage matrix was structurally contiguous with native and grafted cartilage when tested in an osteochondral explant model in vivo. These results demonstrate that this combined approach of a cell homing factor and supportive matrix will promote cell-mediated integrative cartilage repair and improve clinical outcomes of cartilage grafts in the treatment of osteoarthritis., (© 2019 New York Academy of Sciences.)

Recent studies have demonstrated matrix-mineral alterations in bone tissue surrounding osteocytes in estrogen-deficient animals. While cortical bone porosity has been shown to be a contributor to the mechanical properties of bone tissue, little analysis has been done to investigate the effects of estrogen deficiency on bone's microporosities, including the vascular and osteocyte lacunar porosities. In this study we examined alterations in cortical bone microporosity, mineralization, and cancellous bone architecture due to estrogen deficiency in the ovariectomized rat model of postmenopausal osteoporosis. Twenty-week-old female Sprague-Dawley rats were subjected to either ovariectomy or sham surgery. Six weeks post-surgery tibiae were analyzed using high-resolution micro-CT, backscattered electron imaging, nanoindentation, and dynamic histomorphometry. Estrogen deficiency caused an increase in cortical bone vascular porosity, with enlarged vascular pores and little change in tissue mineral density in the proximal tibial metaphysis. Measurements of cancellous architecture corresponded to previous studies reporting a decrease in bone volume fraction, an increase in trabecular separation, and a decrease in trabecular number in the proximal tibia due to estrogen deficiency. Nanoindentation results showed no differences in matrix stiffness in osteocyte-rich areas of the proximal tibia of estrogen-deficient rats, and bone labeling and backscattered electron imaging showed no significant changes in mineralization around the vascular pores. The findings demonstrate local surface alterations of vascular pores due to estrogen deficiency. An increase in cortical vascular porosity may diminish bone strength as well as alter bone mechanotransduction via interstitial fluid flow, both of which could contribute to bone fragility during postmenopausal osteoporosis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The enthesis, or interface between bone and soft tissues such as ligament and tendon, is prone to injury and often does not heal, even post surgical intervention. Interface tissue engineering represents an integrative strategy for regenerating the native enthesis by functionally connecting soft and hard tissues and thereby improving clinical outcome. This review focuses on integrative and cell-instructive scaffold designs that target the healing of the two most commonly injured soft tissue-bone junctions: tendon-bone interface (e.g., rotator cuff) and ligament-bone interface (e.g., anterior cruciate ligament). The inherent connectivity between soft and hard tissues is instrumental for musculoskeletal motion and is therefore a key design criterion for soft tissue regeneration. To this end, scaffold design for soft tissue regeneration have progressed from single tissue systems to the emerging focus on pre-integrated and functional composite tissue units. Specifically, a multifaceted, bioinspired approach has been pursued wherein scaffolds are tailored to stimulate relevant cell responses using spatially patterned structural and chemical cues, growth factors, and/or mechanical stimulation. Moreover, current efforts to elucidate the essential scaffold design criteria via strategic biomimicry are emphasized as these will reduce complexity in composite tissue regeneration and ease the related burden for clinical translation. These innovative studies underscore the clinical relevance of engineering connective tissue integration and have broader impact in the formation of complex tissues and total joint regeneration. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1069-1077, 2018., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

There remains a lack of understanding of the structural changes that occur across the complex, multitissue anterior cruciate ligament (ACL)-to-bone insertion as a function of aging. The objective of this study is to provide a multiscale comparison of matrix properties across the skeletally immature and mature ACL-to-bone insertion. Using complementary imaging methods, micro- and ultrastructural analysis of the insertion revealed that collagen fiber orientation at the interface changes with age, though the degree of collagen organization is maintained over time. These changes are accompanied by a decrease in collagen fibril density and are likely driven by physiological loading. Mineral crystal structure and crystallinity are conserved over time, despite regional differences in crystallinity between the interface and bone. This suggests that mineral chemistry is established early in development and underscores its important functional role. Collectively, these findings provide new insights into interface development and set critical design benchmarks for integrative soft tissue repair.

The anterior cruciate ligament (ACL)-to-bone interface constitutes a complex, multi-tissue structure comprised of contiguous ligament, non-mineralized fibrocartilage, mineralized fibrocartilage, and bone regions. This composite structure enables load transfer between structurally and functionally dissimilar tissues and is critical for ligament homeostasis and joint stability. Presently, there is a lack of quantitative understanding of the matrix composition and organization across this junction, especially after the onset of skeletal maturity. The objective of this study is to characterize the adult bovine ACL-to-bone interface using Fourier transform infrared spectroscopic imaging (FTIRI), testing the hypothesis that regional changes in collagen, proteoglycan, and mineral distribution, as well as matrix organization, persist at the mature insertion. It was observed that while collagen content increases continuously across the adult interface, collagen alignment decreases between ligament and bone. Proteoglycans were primarily localized to the fibrocartilage region and an exponential increase in mineral content was observed between the non-mineralized and mineralized regions. These observations reveal significant changes in collagen distribution and alignment with maturity, and these trends underscore the role of physiologic loading in postnatal matrix remodeling. Findings from this study provide new insights into interface organization and serve as benchmark design criteria for interface regeneration and integrative soft tissue repair. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2513-2523, 2017., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

In this article, we report on the differences in the healing biology of biceps tenodesis performed on either bone or soft tissue in a rat model. This work provides further insight into what may be the optimal strategy for managing biceps-labrum complex disease., Competing Interests: Authors’ Disclosure Statement: The authors report no actual or potential conflict of interest in relation to this article.

Rett syndrome (RTT) is a neurodevelopmental disorder predominately affecting young females, caused by deficiency of the global transcriptional protein methyl CpG binding protein 2 (MeCP2). Osteoblasts express MeCP2 and girls with RTT experience early onset osteoporosis, decreased bone mass and an increased fracture risk. There is no defined treatment for osteoporosis associated with RTT. The present study evaluated the effects of zoledronic acid (ZA), a third generation nitrogen-containing bisphosphonate with primarily anti-osteoclastic activity, in a mouse model of MeCP2 deficiency. Mice received weekly injections of 20μg/kg ZA for six weeks. Due to the shortened lifespan of hemizygous male (Mecp2-null) mice, treatment began at 3weeks of age for this group and corresponding wildtype (WT) male mice. Treatment for heterozygous (HET) and WT female mice began at 8weeks of age. Micro-computed tomography (micro-CT) and dynamic analyses of bone turnover were performed. ZA treatment led to significant increases in bone volume fraction, number, connectivity density and apparent density of trabecular bone in all genotypes of mice. In contrast, cortical bone generally was unaffected by ZA injections. Parameters of bone turnover, including mineral apposition rate, labeled bone surface and bone formation rate decreased after treatment with ZA. Mecp2-null mice had reduced labeled bone surface and bone formation rate compared to WT male mice. The results indicate that ZA treatment significantly improved trabecular bone mass in a murine model of RTT with little effect on cortical bone., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Rotator cuff (RC) tears represent a large proportion of musculoskeletal injuries attended to at the clinic and thereby make RC repair surgeries one of the most widely performed musculoskeletal procedures. Despite the high incidence rate of RC tears, operative treatments have provided minimal functional gains and suffer from high re-tear rates. The hypocellular nature of tendon tissue poses a limited capacity for regeneration. In recent years, great strides have been made in the area of tendonogenesis and differentiation towards tendon cells due to a greater understanding of the tendon stem cell niche, development of advanced materials, improved scaffold fabrication techniques, and delineation of the phenotype development process. Though in vitro models for tendonogenesis have shown promising results, in vivo models have been less successful. The present work investigates structured matrices mimicking the tendon microenvironment as cell delivery vehicles in a rat RC tear model. RC injuries augmented with a matrix delivering rat mesenchymal stem cells (rMSCs) showed enhanced regeneration over suture repair alone or repair with augmentation, at 6 and 12-weeks post-surgery. The local delivery of rMSCs led to increased mechanical properties and improved tissue morphology. We hypothesize that the mesenchymal stem cells function to modulate the local immune and bioactivity environment through autocrine/paracrine and/or cell homing mechanisms. This study provides evidence for improved tendon healing with biomimetic matrices and delivered MSCs with the potential for translation to larger, clinical animal models. The enhanced regenerative healing response with stem cell delivering biomimetic matrices may represent a new treatment paradigm for massive RC tendon tears.

Background: The US education system must find creative and effective ways to foster the healthy development of the approximately 2 million newly arrived immigrant and refugee adolescents, many of whom contend with language barriers, limited prior education, trauma, and discrimination. We identify research priorities for promoting the school success of these youth., Methods: The study used the 4-phase priority-setting method of the Child Health and Nutrition Research Initiative. In the final stage, 132 researchers, service providers, educators, and policymakers based in the United States were asked to rate the importance of 36 research options., Results: The highest priority research options (range 1 to 5) were: evaluating newcomer programs (mean = 4.44, SD = 0.55), identifying how family and community stressors affect newly arrived immigrant and refugee adolescents' functioning in school (mean = 4.40, SD = 0.56), identifying teachers' major stressors in working with this population (mean = 4.36, SD = 0.72), and identifying how to engage immigrant and refugee families in their children's education (mean = 4.35, SD = 0.62)., Conclusion: These research priorities emphasize the generation of practical knowledge that could translate to immediate, tangible benefits for schools. Funders, schools, and researchers can use these research priorities to guide research for the highest benefit of schools and the newly arrived immigrant and refugee adolescents they serve., (© 2017, American School Health Association.)

Growth delay is common in children with chronic kidney disease (CKD), often associated with poor quality of life. The role of anemia in uremic growth delay is poorly understood. Here we describe an induction of uremic growth retardation by a 0.2% adenine diet in wild-type (WT) and hepcidin gene (Hamp) knockout (KO) mice, compared with their respective littermates fed a regular diet. Experiments were started at weaning (3 wk). After 8 wk, blood was collected and mice were euthanized. Adenine-fed WT mice developed CKD (blood urea nitrogen 82.8 ± 11.6 mg/dl and creatinine 0.57 ± 0.07 mg/dl) and were 2.1 cm shorter compared with WT controls. WT adenine-fed mice were anemic and had low serum iron, elevated Hamp, and elevated IL6 and TNF-α. WT adenine-fed mice had advanced mineral bone disease (serum phosphorus 16.9 ± 3.1 mg/dl and FGF23 204.0 ± 115.0 ng/ml) with loss of cortical and trabecular bone volume seen on microcomputed tomography. Hamp disruption rescued the anemia phenotype resulting in improved growth rate in mice with CKD, thus providing direct experimental evidence of the relationship between Hamp pathway and growth impairment in CKD. Hamp disruption ameliorated CKD-induced growth hormone-insulin-like growth factor 1 axis derangements and growth plate alterations. Disruption of Hamp did not mitigate the development of uremia, inflammation, and mineral and bone disease in this model. Taken together, these results indicate that an adenine diet can be successfully used to study growth in mice with CKD. Hepcidin appears to be related to pathways of growth retardation in CKD suggesting that investigation of hepcidin-lowering therapies in juvenile CKD is warranted., (Copyright © 2016 the American Physiological Society.)

This study assessed the association between exposure to political violence over a 25-year period and adult functioning among a population that has experienced protracted and severe political conflict. Instead of aggregating exposure to political violence across time and type of exposure, as is commonly done, the event history calendar pioneered in this study assessed exposure to five forms of political violence annually from 1987 to 2011 in a representative sample of 1788 adults, aged 37 on average, in the occupied Palestinian territories (West Bank, East Jerusalem, and the Gaza Strip). This method allowed for the identification of trajectories of exposure to political violence from childhood to adulthood using latent profile analysis. We then correlated the trajectories of exposure to measures of economic, political, community, family, psychological, and health functioning. As expected, being shot at, having one's home raided, being hit or kicked, being verbally abused, and witnessing someone close being humiliated were all elevated during periods of heightened political conflict (the first intifada (1987-1993) and, less so, the second intifada (2000-2005)). In addition, 12% of women and men reported high and persistent levels of exposure to humiliation (being verbally abused and/or witnessing someone close being humiliated) across the entire 25-year period. These individuals lived predominantly in neighborhoods with a high Israeli military presence. Compared to those who experienced periodic exposure to political violence, persistently humiliated men and women reported significantly lower health, economic, political, and psychological functioning, as well as higher social cohesion and political expression. Relevant literatures are reviewed when concluding that persistent humiliation is a neglected form of political violence that is best represented as a direct (versus structural), acute (versus chronic), macro (versus micro), and high-grade (versus low-grade) stressor whose particular injury is due to the violation of individual and collective identity, rights, justice and dignity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder due to mutations affecting the neural transcription factor MeCP2. Approximately 50% of affected females have decreased bone mass. We studied osteoblast function using a murine model of RTT. Female heterozygote (HET) and male Mecp2-null mice were compared to wild type (WT) mice. Micro-CT of tibia from 5 week-old Mecp2-null mice showed significant alterations in trabecular bone including reductions in bone volume fraction (-29%), number (-19%), thickness (-9%) and connectivity density (-32%), and increases in trabecular separation (+28%) compared to WT. We also found significant reductions in cortical bone thickness (-18%) and in polar moment of inertia (-45%). In contrast, cortical and trabecular bone from 8 week-old WT and HET female mice were not significantly different. However, mineral apposition rate, mineralizing surface and bone formation rate/bone surface were each decreased in HET and Mecp2-null mice compared to WT mice. Histomorphometric analysis of femurs showed decreased numbers of osteoblasts but similar numbers of osteoclasts compared to WT, altered osteoblast morphology and decreased tissue synthesis of alkaline phosphatase in Mecp2-null and HET mice. Osteoblasts cultured from Mecp2-null mice, which unlike WT osteoblasts did not express MeCP2, had increased growth rates, but reductions in mRNA expression of type I collagen, Runx2 and Osterix compared to WT osteoblasts. These results indicate that MeCP2 deficiency leads to altered bone growth. Osteoblast dysfunction was more marked in Mecp2-null male than in HET female mice, suggesting that expression of MeCP2 plays a critical role in bone development., (Copyright © 2015. Published by Elsevier Inc.)

With greater than 500,000 orthopaedic procedures performed in the United States each year requiring a bone graft, the development of novel graft materials is necessary. We report that some porous polymer/ceramic composite scaffolds possess intrinsic osteoinductivity as shown through their capacity to induce in vivo host osteoid mineralization and in vitro stem cell osteogenesis making them attractive synthetic bone graft substitutes. It was discovered that certain low crystallinity ceramics partially dissociate into simple signaling molecules (i.e., calcium and phosphate ions) that induce stem cells to endogenously produce their own osteoinductive proteins. Review of the literature has uncovered a variety of simple signaling molecules (i.e., gases, ions, and redox reagents) capable of inducing other desirable stem cell differentiation through endogenous growth factor production. Inductive simple signaling molecules, which we have termed inducerons, represent a paradigm shift in the field of regenerative engineering where they can be utilized in place of recombinant protein growth factors.

A number of studies have focused on the beneficial properties of Curcumin (diferuloyl methane, used in South Asian cuisine and traditional medicine) such as the chemoprevention of cancer. Recent studies have also indicated that this material has significant benefits for the treatment of cancer and is currently undergoing several clinical trials. We have been interested in the application of this compound as a therapeutic agent for advanced prostate cancer, particularly the skeletal complications in this malignancy. Our earlier work indicated that this compound could inhibit the osteomimetic properties which occur in castration resistant prostate cancer cells, by interfering with the common denominators between these cancer cells and the bone cells in the metastatic tumor microenvironment, namely the osteoblasts and the osteoclast. We predicted that curcumin could break the vicious cycle of reciprocal stimulation that results in uncontrolled osteolysis in the bony matrix. In this work, we have evaluated the potential of this compound in inhibiting the bone metastasis of hormone refractory prostate cancer cells in an established animal model. Our results strongly suggest that curcumin modulates the TGF- β signaling that occurs due to bone matrix degradation by up-regulating the metastasis inhibitory bone morphogenic protein-7 (BMP- 7). This enhancement of BMP-7 in the context of TGF- β in the tumor microenvironment is shown to enhance the mesenchymal-to-epithelial transition. Most importantly, we show that as a result of BMP-7 up-regulation, a novel brown/beige adipogenic differentiation program is also up-regu- lated which plays a role in the inhibition of bone metastasis. Our results suggest that curcumin may subvert the TGF- β signaling to an alternative adipogenic differentiation program in addition to the previously established interference with the osteomimetic properties, thus inhibiting the bone metastatic processes in a chemopreventive as well as therapeutic setting.

To test if osteoporosis alters mechanical load-induced interstitial fluid flow in bone, this study examined the combined effect of estrogen deficiency and external loading on solute transport around osteocytes. An in vivo tracer, FITC-labeled bovine serum albumin, was injected into anesthetized ovariectomized and control female Sprague-Dawley rats before the right tibia was subjected to a controlled, physiological, non-invasive sinusoidal load to mimic walking. Tracer movement through the lacunar-canalicular system surrounding osteocytes was quantified in cortical and cancellous bone from the proximal tibia using confocal microscopy, with the non-loaded tibia serving as internal control. Overall, the application of mechanical loading increased the percentage of osteocyte lacunae labeled with injected tracer, and ovariectomy further enhanced movement of tracer. An analysis of separate regions demonstrated that ovariectomy enhanced in vivo transport of the injected tracer in the cancellous bone of the tibial epiphysis and metaphysis but not in the cortical bone of the metaphysis. These findings show that bone changes due to reduced estrogen levels alter convectional transport around osteocytes in cancellous bone and demonstrate a functional difference of interstitial fluid flow around osteocytes in estrogen-deficient rats undergoing the same physical activity as controls. The altered interstitial fluid flow around osteocytes is likely related to nanostructural matrix-mineral level differences recently demonstrated at the lacunar-canalicular surface of estrogen-deficient rats, which could affect the transmission of mechanical loads to the osteocyte., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Current micro-computed tomography (µCT) systems allow scanning bone at resolutions capable of three-dimensional (3D) characterization of intracortical vascular porosity and osteocyte lacunae. However, the scanning and reconstruction parameters along with the image segmentation method affect the accuracy of the measurements. In this study, the effects of scanning resolution and image threshold method in quantifying small features of cortical bone (vascular porosity, vascular canal diameter and separation, lacunar porosity and density, and tissue mineral density) were analyzed. Cortical bone from the tibia of Sprague-Dawley rats was scanned at 1-µm and 4-µm resolution, reconstructions were density-calibrated, and volumes of interest were segmented using approaches based on edge-detection or histogram analysis. In 1-µm resolution scans, the osteocyte lacunar spaces could be visualized, and it was possible to separate the lacunar porosity from the vascular porosity. At 4-µm resolution, the vascular porosity and vascular canal diameter were underestimated, and osteocyte lacunae were not effectively detected, whereas the vascular canal separation and tissue mineral density were overestimated compared to 1-µm resolution. Resolution had a much greater effect on the measurements than did threshold method, showing partial volume effects at resolutions coarser than 2 µm in two separate analyses, one of which assessed the effect of resolution on an object of known size with similar architecture to a vascular pore. Although there was little difference when using the edge-detection versus histogram-based threshold approaches, edge-detection was somewhat more effective in delineating canal architecture at finer resolutions (1-2 µm). In addition, use of a high-resolution (1 µm) density-based threshold on lower resolution (4 µm) density-calibrated images was not effective in improving the lower-resolution measurements. In conclusion, if measuring cortical vascular microarchitecture, especially in small animals, a µCT resolution of 1 to 2 µm is appropriate, whereas a resolution of at least 1 µm is necessary when assessing osteocyte lacunar porosity., (© 2014 American Society for Bone and Mineral Research.)

It is believed that orthopedic and implant longevity can be improved by optimizing fixation, or direct bone-implant contact, through the stimulation of new bone formation around the implant. The purpose of this study was to determine whether heat (600°C) or radiofrequency plasma glow discharge (RFGD) pretreatment of Ti6Al4V stimulated calcium-phosphate mineral formation in cultures of attached MC3T3 osteoprogenitor cells with or without a fibronectin coating. Calcium-phosphate mineral was analyzed by flame atomic absorption spectrophotometry, scanning electron microscopy (SEM)/electron dispersive X-ray microanalysis (EDAX) and Fourier transformed infrared spectroscopy (FTIR). RFGD and heat pretreatments produced a general pattern of increased total soluble calcium levels, although the effect of heat pretreatment was greater than that of RFGD. SEM/EDAX showed the presence of calcium-and phosphorus-containing particles on untreated and treated disks that were more numerous on fibronectin-coated disks. These particles were observed earliest (1 week) on RFGD-pretreated surfaces. FTIR analyses showed that the heat pretreatment produced a general pattern of increased levels of apatite mineral at 2-4 weeks; a greater effect was observed for fibronectin-coated disks compared to uncoated disks. The observed findings suggest that heat pretreatment of Ti6Al4V increased the total mass of the mineral formed in MC3T3 osteoprogenitor cell cultures more than RFGD while the latter pretreatment hastened the early deposition of mineral. These findings help to support the hypothesis that the pretreatments enhance the osteoinductive properties of the alloy., (Copyright © 2013 Wiley Periodicals, Inc.)

While reduced estrogen levels have been shown to increase bone turnover and induce bone loss, there has been little analysis of the effects of diminished estrogen levels on the lacunar-canalicular porosity that houses the osteocytes. Alterations in the osteocyte lacunar-canalicular microenvironment may affect the osteocyte's ability to sense and translate mechanical signals, possibly contributing to bone degradation during osteoporosis. To investigate whether reduced estrogen levels affect the osteocyte microenvironment, this study used high-resolution microscopy techniques to assess the lacunar-canalicular microstructure in the rat ovariectomy (OVX) model of postmenopausal osteoporosis. Confocal microscopy analyses indicated that OVX rats had a larger effective lacunar-canalicular porosity surrounding osteocytes in both cortical and cancellous bone from the proximal tibial metaphysis, with little change in cortical bone from the diaphysis or cancellous bone from the epiphysis. The increase in the effective lacunar-canalicular porosity in the tibial metaphysis was not due to changes in osteocyte lacunar density, lacunar size, or the number of canaliculi per lacuna. Instead, the effective canalicular size measured using a small molecular weight tracer was larger in OVX rats compared to controls. Further analysis using scanning and transmission electron microscopy demonstrated that the larger effective canalicular size in the estrogen-deficient state was due to nanostructural matrix-mineral level differences like loose collagen surrounding osteocyte canaliculi. These matrix-mineral differences were also found in osteocyte lacunae in OVX, but the small surface changes did not significantly increase the effective lacunar size. The alterations in the lacunar-canalicular surface mineral or matrix environment appear to make OVX bone tissue more permeable to small molecules, potentially altering interstitial fluid flow around osteocytes during mechanical loading., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Evaluation of the skin phenotype in osteogenesis imperfecta (OI) typically involves biochemical measurements, such as histologic or biochemical assessment of the collagen produced from biopsy-derived dermal fibroblasts. As an alternative, the current study utilized non-invasive magnetic resonance imaging (MRI) microscopy and optical spectroscopy to define biophysical characteristics of skin in an animal model of OI. MRI of skin harvested from control, homozygous oim/oim and heterozygous oim/+ mice demonstrated several differences in anatomic and biophysical properties. Fourier transform infrared imaging spectroscopy (FT-IRIS) was used to interpret observed MRI signal characteristics in terms of chemical composition. Differences between wild-type and OI mouse skin included the appearance of a collagen-depleted lower dermal layer containing prominent hair follicles in the oim/oim mice, accounting for 55% of skin thickness in these. The MRI magnetization transfer rate was lower by 50% in this layer as compared to the upper dermis, consistent with lower collagen content. The MRI transverse relaxation time, T2, was greater by 30% in the dermis of the oim/oim mice compared to controls, consistent with a more highly hydrated collagen network. Similarly, an FT-IRIS-defined measure of collagen integrity was 30% lower in the oim/oim mice. We conclude that characterization of phenotypic differences between the skin of OI and wild-type mice by MRI and FT-IRIS is feasible, and that these techniques provide powerful complementary approaches for the analysis of the skin phenotype in animal models of disease., (Copyright © 2011 John Wiley & Sons, Ltd.)

The noninvasive early detection of specific matrix alterations in degenerative cartilage disease would be of substantial use in basic science studies and clinically, but remains an elusive goal. Recently developed MRI methods exhibit some specificity, but require contrast agents or nonstandard pulse sequences and hardware. We present a multiexponential approach which does not require contrast agents or specialized hardware, and uses a standard multiple-echo spin-echo sequence. Experiments were performed on tissue models of degenerative cartilage using enzymes with distinct actions. MR results were validated using histologic, biochemical and infrared spectroscopic analyses. The sulfated glycosaminoglycan per dry weight (dw) in bovine nasal cartilage was 0.72 ± 0.06 mg/mg dw and was reduced through chondroitinase AC and collagenase digestion to 0.56 ± 0.12 and 0.58 ± 0.13 mg/mg dw, respectively. Multiexponential analysis of data obtained at 9.4 T permitted the identification of tissue compartments assigned to the proteoglycan component of the matrix and to bulk water. Enzymatic treatment resulted in a significant reduction in the ratio of proteoglycan-bound to free water from 0.13 ± 0.02 in control cartilage to 0.03 ± 0.02 and 0.05 ± 0.06 under chondroitinase AC and collagenase treatment, respectively. As expected, monoexponential T(2) increased with both degradation protocols, but without further specificity to the nature of the degradation. An important eventual extension of this approach may be to map articular cartilage degeneration in the clinical setting. As an initial step towards this, localized multiexponential T(2) analysis was performed on control and trypsin treated excised bovine patella. The results obtained on this articular cartilage sample were readily interpretable in terms of proteoglycan-associated and relatively free water compartments. In potential clinical applications, signal-to-noise ratio constraints will define the threshold for the detection of macromolecular compartment changes at a given spatial scale. The multiexponential approach has potential application to the early detection of cartilage degradation with the use of appropriate pulse parameters under high signal-to-noise ratio conditions., (Copyright © 2011 John Wiley & Sons, Ltd.)

Transcription factors that play a role in ossification during development are expected to participate in postnatal fracture repair since the endochondral bone formation that occurs in embryos is recapitulated during fracture repair. However, inherent differences exist between bone development and fracture repair, including a sudden disruption of tissue integrity followed by an inflammatory response. This raises the possibility that repair-specific transcription factors participate in bone healing. Here, we assessed the consequence of loss of early growth response gene 1 (EGR-1) on endochondral bone healing because this transcription factor has been shown to modulate repair in vascularized tissues. Model fractures were created in ribs of wild type (wt) and EGR-1(-/-) mice. Differences in tissue morphology and composition between these two animal groups were followed over 28 post fracture days (PFDs). In wt mice, bone healing occurred in healing phases characteristic of endochondral bone repair. A similar healing sequence was observed in EGR-1(-/-) mice but was impaired by alterations. A persistent accumulation of fibrin between the disconnected bones was observed on PFD7 and remained pronounced in the callus on PFD14. Additionally, the PFD14 callus was abnormally enlarged and showed increased deposition of mineralized tissue. Cartilage ossification in the callus was associated with hyper-vascularity and -proliferation. Moreover, cell deposits located in proximity to the callus within skeletal muscle were detected on PFD14. Despite these impairments, repair in EGR-1(-/-) callus advanced on PFD28, suggesting EGR-1 is not essential for healing. Together, this study provides genetic evidence that EGR-1 is a pleiotropic regulator of endochondral fracture repair., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Cysteine (C)-X-C motif chemokine receptor 4 (CXCR4), the primary receptor for stromal cell-derived factor-1 (SDF-1), is involved in bone morphogenic protein 2 (BMP2)-induced osteogenic differentiation of mesenchymal progenitors. To target the in vivo function of CXCR4 in bone and explore the underlying mechanisms, we conditionally inactivated CXCR4 in osteoprecursors by crossing osterix (Osx)-Cre mice with floxed CXCR4 (CXCR4(fl/fl)) mice to generate knock-outs with CXCR4 deletion driven by the Osx promoter (Osx::CXCR4(fl/fl)). The Cre-mediated excision of CXCR4 occurred exclusively in bone of Osx::CXCR4(fl/fl) mice. When compared with littermate controls, Osx::CXCR4(fl/fl) mice developed smaller osteopenic skeletons as evidenced by reduced trabecular and cortical bone mass, lower bone mineral density, and a slower mineral apposition rate. In addition, Osx::CXCR4(fl/fl) mice displayed chondrocyte disorganization in the epiphyseal growth plate associated with decreased proliferation and collagen matrix syntheses. Moreover, mature osteoblast-related expression of type I collagen α1 and osteocalcin was reduced in bone of Osx::CXCR4(fl/fl) mice versus controls, suggesting that CXCR4 deficiency results in arrested osteoblast progression. Primary cultures for osteoblastic cells derived from Osx::CXCR4(fl/fl) mice also showed decreased proliferation and impaired osteoblast differentiation in response to BMP2 or BMP6 stimulation, and suppressed activation of intracellular BMP receptor-regulated Smads (R-Smads) and Erk1/2 was identified in CXCR4-deficient cells and bone tissues. These findings provide the first in vivo evidence that CXCR4 functions in postnatal bone development by regulating osteoblast development in cooperation with BMP signaling. Thus, CXCR4 acts as an endogenous signaling component necessary for bone formation.

Purpose: The objective of this study was to assess the performance of a degradable porous polyurethane scaffold in a partial meniscectomy ovine model., Methods: We subjected 42 skeletally mature ewes to unilateral partial excision of the lateral meniscus. In 19 animals the defect was left unfilled; in 23 animals a scaffold was inserted. Knees were examined by magnetic resonance imaging, gross inspection, and histologic inspection of the cartilage of the tibial plateau., Results: In contrast to what has been previously reported in a complete meniscal replacement model, cartilage damage did not occur under the site of scaffold implantation; this was likely influenced by the rapid infiltration of cells and the dense tissue that formed within the scaffold. Cartilage damage in both groups was located close to the midline of the joint. No significant difference in the condition of the articular cartilage of the tibial plateau was seen between groups up to 12 months postoperatively. This result was influenced by the fact that the partly meniscectomized knees also showed unexpected tissue regeneration within the defect site, which raises concern about the suitability of using a partial meniscectomy as a control in the ovine model., Conclusions: Our study has shown that implantation of a polyurethane scaffold in a partial meniscectomy ovine model promotes tissue ingrowth without damaging the cartilage with which it articulates., Clinical Relevance: Meniscal deficiency is a common occurrence, the effective clinical management of which is limited by the absence of an off-the-shelf implantable construct., (Copyright © 2010 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

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.

Porous polyurethane foams (PUR) have been extensively evaluated as meniscal replacement materials and show great promise enabling infiltration of cells and fibrocartilage formation in vivo. Similar to most materials, PUR demonstrates progressive degeneration of opposing cartilage; however, the damage mechanism is impossible to determine because no information exists on the frictional properties of PUR-cartilage interfaces. The goals of this study were to characterize the frictional behavior of a cartilage-PUR interface across a range of articulating conditions and assess the resulting morphological changes to the cartilage surface following articulation. Articular cartilage was oscillated against PUR or stainless steel using phosphate-buffered saline (PBS) and synovial fluid as lubricants. Following friction testing, cartilage and PUR samples were analyzed with environmental scanning electron microscopy and histological staining to determine changes in tissue morphology. Stribeck-surface analysis demonstrated distinct lubrication modes; however, boundary mode lubrication was dominant in cartilage-PUR interfaces and the low-friction pressure-borne lubrication mechanism present in native joints was absent. Microscopy noted obvious wear, with disruption of the collagen architecture and concomitant proteoglycan loss in cartilage articulated against PUR. These data collectively point to the importance of frictional properties as design parameters for implants and materials for soft tissue replacement., (Published by Wiley Periodicals, Inc. J Orthop Res 28:1292-1299, 2010.)

Natural polymer chitosan and synthetic polymer poly(lactide-co-glycolide) (PLAGA) have been investigated for a variety of tissue engineering applications. We have previously reported the fabrication and in vitro evaluation of a novel chitosan/PLAGA sintered microsphere scaffold for load-bearing bone tissue engineering applications. In this study, the in vitro degradation characteristics of the chitosan/PLAGA scaffold and the in vivo bone formation capacity of the chitosan/PLAGA-based scaffolds in a rabbit ulnar critical-sized-defect model were investigated. The chitosan/PLAGA scaffold showed slower degradation than the PLAGA scaffold in vitro. Although chitosan/PLAGA scaffold showed a gradual decrease in compressive properties during the 12-week degradation period, the compressive strength and compressive modulus remained in the range of human trabecular bone. Chitosan/PLAGA-based scaffolds were able to guide bone formation in a rabbit ulnar critical-sized-defect model. Microcomputed tomography analysis demonstrated that successful bridging of the critical-sized defect on the sides both adjacent to and away from the radius occurred using chitosan/PLAGA-based scaffolds. Immobilization of heparin and recombinant human bone morphogenetic protein-2 on the chitosan/PLAGA scaffold surface promoted early bone formation as evidenced by complete bridging of the defect along the radius and significantly enhanced mechanical properties when compared to the chitosan/PLAGA scaffold. Furthermore, histological analysis suggested that chitosan/PLAGA-based scaffolds supported normal bone formation via intramembranous formation., (2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The murine mesenchymal cell line, C3H10T1/2 in micromass culture undergoes chondrogenic differentiation with the addition of BMP-2. This study compares the use of BMP-2 vs. insulin, transferrin, and sodium selenite (ITS) to create a chondrogenic micromass cell culture system that models cartilage calcification in the presence of 4mM inorganic phosphate. BMP-2 treated cultures showed more intense alcian blue staining for proteoglycans than ITS treated cultures at early time points. Both ITS and BMP-2 treated cultures showed similar mineral deposition in cultures treated with 4mM phosphate via von Kossa staining, however FTIR spectroscopy of cultures showed different matrix properties. ITS treated cultures produced matrix that more closely resembled mouse calcified cartilage by FTIR analysis. (45)Ca uptake curves showed delayed onset of mineralization in cultures treated with BMP-2, however they had an increased rate of mineralization (initial slope of (45)Ca uptake curve) when compared to the cultures treated with ITS. Immunohistochemistry showed the presence of both collagens type I and type II in BMP-2 and ITS treated control (1mM inorganic phosphate) and mineralizing cultures. BMP-2 treated mineralizing cultures displayed more intense staining for collagen type II than all other cultures. Collagen type X staining was detected at Day 9 only in mineralizing cultures treated with ITS. Western blotting of Day 9 cultures confirmed the presence of collagen type X in the mineralizing ITS cultures, and also showed very small amounts of collagen type X in BMP-2 treated cultures and control ITS cultures. By Day 16 all cultures stained positive for collagen type X. These data suggest that BMP-2 induces a more chondrogenic phenotype, while ITS treatment favors maturation and hypertrophy of the chondrocytes in the murine micromass cultures.

Intermittent parathyroid hormone administration can enhance fracture healing in an animal model. Despite the success of exogenous parathyroid hormone on fracture healing and spine fusion, few studies have examined the role of parathyroid hormone on cartilage formation. We determined the effects of intermittent parathyroid hormone on cartilage formation in a rabbit microfracture model of cartilage regeneration. Twelve rabbits were divided into three equal groups: (1) microfracture alone, (2) microfracture + parathyroid hormone daily for 7 days, and (3) microfracture + parathyroid hormone for 28 days. Nonoperated contralateral knees were used as controls. The animals were sacrificed at 3 months and gross and histologic analysis was performed. The microfracture alone group demonstrated the most healing on gross and histologic analysis. Treatment with either 1 or 4 weeks of parathyroid hormone inhibited cartilage formation. Although discouraging from a cartilage repair point of view, this study suggests that the role parathyroid hormone administration has in clinical fracture healing must be examined carefully. Although parathyroid hormone is beneficial to promote healing in spine fusion and midshaft fractures, its deleterious effects on cartilage formation suggests that it may have adverse effects on the outcomes of periarticular fractures such as tibial plateau injuries that require cartilage healing for a successful clinical outcome.

Subchondral bone and articular cartilage play complementary roles in load bearing of the joints. Although the biomechanical coupling between subchondral bone and articular cartilage is well established, it remains unclear whether direct biochemical communication exists between them. Previously, the calcified cartilage between these two compartments was generally believed to be impermeable to transport of solutes and gases. However, recent studies found that small molecules could penetrate into the calcified cartilage from the subchondral bone. To quantify the real-time solute transport across the calcified cartilage, we developed a novel imaging method based on fluorescence loss induced by photobleaching (FLIP). Diffusivity of sodium fluorescein (376 Da) was quantified to be 0.07 +/- 0.03 and 0.26 +/- 0.22 microm(2)/s between subchondral bone and calcified cartilage and within the calcified cartilage in the murine distal femur, respectively. Electron microscopy revealed that calcified cartilage matrix contained nonmineralized regions (approximately 22% volume fraction) that are either large patches (53 +/- 18 nm) among the mineral deposits or numerous small regions (4.5 +/- 0.8 nm) within the mineral deposits, which may serve as transport pathways. These results suggest that there exists a possible direct signaling between subchondral bone and articular cartilage, and they form a functional unit with both mechanical and biochemical interactions, which may play a role in the maintenance and degeneration of the joint., ((c) 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Dystrophic calcifications often occur after injury, infection, or onset of certain rheumatic diseases. Treatment has been limited to surgical removal following failure of medical therapy. In an attempt to establish a reproducible animal model for dystrophic calcification that permitted the screening of potential interventions, we evaluated cardiotoxin (injury)-induced calcifications in three murine strains at both the cellular and ultrastructural levels. All osteopontin null mice and tumor necrosis factor receptor null mice on a C57B6 background had calcifications at days 3 and 7 after injury compared to 75% of wild-type C57B6 mice. There was no difference in mineral content among calcifications from the three mouse strains. Osteogenesis was suggested by the expression of osteocalcin, osterix, and alkaline phosphatase in calcified murine muscle tissue. Osteoclast-like cells facilitated the removal of transient dystrophic deposits (<28 days) in all models. However, none of the models showed an association of mineral crystals with collagen, suggesting that the deposits were not bone-like. The dystrophic mechanism was validated as cell death, and mitochondrial calcifications occurred soon after skeletal muscle injury in the three murine strains.

Little is known about osteonal bone mineral and matrix properties, although these properties are of major importance for the understanding of bone alterations related to age and bone diseases such as osteoporosis. During aging, bone undergoes modifications that compromise their structural integrity as shown clinically by the increase of fracture incidence with age. Based on Fourier transform infrared (FTIR) analysis from baboons between 0 and 32 yr of age, consistent systematic variations in bone properties as a function of tissue age are reported within osteons. The patterns observed were independent of animal age and positively correlated with bone tissue elastic behavior measured by nano-indentation. As long as tissue age is expressed as a percentage of the entire osteon radius, osteonal analyses can be used to characterize disease changes independent of the size of the osteon. These mineral and matrix analyses can be used to explain bone fragility. The mineral content (mineral-to-matrix ratio) was correlated with the animal age in both old (interstitial) and newly formed bone tissue, showing for the first time that age-related changes in BMC can be explain by an alteration in the mineralization process itself and not only by an imbalance in the remodeling process.

Bone is a composite porous material with two functional levels of porosity: the vascular porosity that surrounds blood vessels and the lacunar-canalicular porosity that surrounds the osteocytes. Both the vascular porosity and lacunar-canalicular porosity are directly involved in interstitial fluid flow, thought to play an important role in bone's maintenance. Because of the small dimensions of the lacunar-canalicular porosity, interstitial fluid space has been difficult to visualize and quantify. We report a new staining protocol that is reliable and easily reproducible, using fluorescein isothiocyanate (FITC) as a probe visualized by confocal microscopy. Reconstructed FITC-stained cross sections enable effective visualization of bone microstructure and microporosities. This new staining process can be used to analyze interstitial fluid space, providing high-resolution quantification of the vascular pores and the lacunar-canalicular network of cortical and cancellous bone.

Cathepsin K deficiency in humans causes pycnodysostosis, which is characterized by dwarfism and osteosclerosis. Earlier studies of 10-week-old male cathepsin K-deficient (knockout, KO) mice showed their bones were mechanically more brittle, while histomorphometry showed that both osteoclasts and osteoblasts had impaired activity relative to the wild type (WT). Here, we report detailed mineral and matrix analyses of the tibia of these animals based on Fourier transform infrared microspectroscopy and imaging. At 10 weeks, there was significant hypercalcification of the calcified cartilage and cortices in the KO. Carbonate content was elevated in the KO calcified cartilage as well as cortical and cancellous bone areas. These data suggest that cathepsin K does not affect mineral deposition but has a significant effect on mineralized tissue remodeling. Since growth plate abnormalities were extensive despite reported low levels of cathepsin K expression in the calcified cartilage, we used a differentiating chick limb-bud mesenchymal cell system that mimics endochondral ossification but does not contain osteoclasts, to show that cathepsin K inhibition during initial stages of mineral deposition retards the mineralization process while general inhibition of cathepsins can increase mineralization. These data suggest that the hypercalcification of the cathepsin K-deficient growth plate is due to persistence of calcified cartilage and point to a role of cathepsin K in bone tissue development as well as skeletal remodeling.

Objective: It was hypothesized that controlled, scaffold removal in engineered cartilage constructs would improve their collagen content and mechanical properties over time in culture., Design: Preliminary experiments characterized the effects of agarase on cell-free agarose disks and cartilage explants. Immature bovine chondrocytes were encapsulated in agarose, cultured to day 42, and incubated with 100 units/mL agarase for 48 h. After treatment, constructs were cultured to day 91. The compressive Young's modulus and dynamic modulus of the constructs were determined every 2 weeks and immediately after agarase treatment. Post-mechanical testing, constructs were processed for biochemistry and histology., Results: Agarase treatment on explants had no detrimental effect on the cartilage matrix. Treatment applied to engineered constructs on day 42 did not affect DNA or collagen content. Agarase treatment decreased tissue GAG content (via GAG loss to the media) and Young's modulus, both of which recovered to control values over time in culture. By day 91 agarase-treated constructs possessed approximately 25% more DNA, approximately 60% more collagen, and approximately 40% higher dynamic modulus compared to untreated controls., Conclusions: Scaffold degradation increased construct collagen content and dynamic mechanical properties, affirming the experimental hypothesis. The mechanism may lie in increased nutrient transport, increased space for collagen fibril formation, and cellular response to the loss of GAG with agarase treatment. The results highlight the role of the scaffold in retaining synthesized matrix during early and late tissue formation. This work also shows promise in developing an engineered tissue that may be completely free of scaffold material for clinical implantation.

Study Design: The posterolateral rabbit spinal fusion model was used to assess the effect of intermittent parathyroid hormone on spinal fusion outcomes., Objective: To test the hypothesis that intermittent parathyroid hormone (PTH) improves spinal fusion outcomes in the rabbit posterolateral spinal fusion model., Summary of Background Data: Spinal fusion is the definitive management for spinal deformity or instability, yet despite current technology, 5% to 40% of lumbar fusions result in pseudarthrosis. Animal studies have demonstrated enhanced fracture healing with the use of PTH, but the effect of PTH on spinal fusion is poorly described., Methods: Forty-four male New Zealand white rabbits underwent bilateral posterolateral spine fusion (L5-L6 level). Twenty-two rabbits received daily subcutaneous injections of PTH (1-34) (10 microg/kg) and 22 received an injection of saline fluid. All were killed 6 weeks after surgery. L5-L6 vertebral segments were removed and analyzed with manual bending, faxitron radiography, microCT, and histomorphometry., Results: Manual bending identified fusion in 30% (control) versus 81% (PTH) animals (P < 0.001). A radiographic scoring system ("0" = no bone formation, "5" = full fusion) resulted in an average score of 3.36 (control) versus 4.51 (PTH) (P < 0.001). MicroCT analysis demonstrated a median mass of 3.5 cc (control) (range, 2.25-5.40 cc) versus 6.03 cc (PTH) (range, 4.34-10.58 cc) (P < 0.001). Histology showed a median percentage bone area of 14.3% (control) (n = 12) versus 29.9% (PTH) (n = 15) (P < 0.001). The median percentage cartilage was 2.7% (control) (n = 5) versus 26.6% (PTH) (n = 5) (P < 0.01). Osteoclast quantification revealed median values of 140.5 (control) (n = 6) and 345.0 (PTH) (n = 8) (P < 0.001) respectively, and the percentage of osteoblasts revealed a median value of 31.4% (control) (n = 6) versus 64.4% (PTH) (n = 8) (P < 0.001)., Conclusion: Intermittent PTH administration increased posterolateral fusion success in rabbits. Fusion bone mass and histologic determinants were also improved with PTH treatment. PTH has promise for use as an adjunctive agent to improve spinal fusion in clinical medicine.

The debilitating effects of rotator cuff tears and the high incidence of failure associated with current grafts underscore the clinical demand for functional solutions for tendon repair and augmentation. To address this challenge, we have designed a poly(lactide-co-glycolide) (PLGA) nanofiber-based scaffold for rotator cuff tendon tissue engineering. In addition to scaffold design and characterization, the objective of this study was to evaluate the attachment, alignment, gene expression, and matrix elaboration of human rotator cuff fibroblasts on aligned and unaligned PLGA nanofiber scaffolds. Additionally, the effects of in vitro culture on scaffold mechanical properties were determined over time. It has been hypothesized that nanofiber organization regulates cellular response and scaffold properties. It was observed that rotator cuff fibroblasts cultured on the aligned scaffolds attached along the nanofiber long axis, whereas the cells on the unaligned scaffold were polygonal and randomly oriented. Moreover, distinct integrin expression profiles on these two substrates were observed. Quantitative analysis revealed that cell alignment, distribution, and matrix deposition conformed to nanofiber organization and that the observed differences were maintained over time. Mechanical properties of the aligned nanofiber scaffolds were significantly higher than those of the unaligned, and although the scaffolds degraded in vitro, physiologically relevant mechanical properties were maintained. These observations demonstrate the potential of the PLGA nanofiber-based scaffold system for functional rotator cuff repair. Moreover, nanofiber organization has a profound effect on cellular response and matrix properties, and it is a critical parameter for scaffold design.

Rapid implant fixation could prove beneficial in a host of clinical applications from total joint arthroplasty to trauma. We hypothesized that a novel self-assembled monolayer of phosphonate molecules (SAMP) covalently bonded to the oxide surface of titanium alloy would enhance bony integration. Beaded metallic rods were treated with one of three coatings: SAMP, SAMP + RGD peptide, or hydroxyapatite. Rods were inserted retrogradely into both distal femurs of 60 rabbits. Fifteen rabbits were sacrificed at 2, 4, 8, and 16 weeks. At each time, seven specimens for mechanical pull-out testing and three for histomorphometric analysis were available for each coating. At four weeks, both SAMP groups had significantly higher failure loads when compared to hydroxyapatite (p < 0.01). No significant differences were found among groups at other times, though the SAMP-alone group remained stronger at 16 weeks. Histology showed abundant new bone formation around all the three groups, though more enhanced formation was apparent in the two SAMP groups. With this novel treatment, with or without RGD, the failure load of implants doubled in half the time as compared with hydroxyapatite. Where early implant fixation is important, the SAMP treatment provides a simple, cost-effective enhancement to bony integration of orthopaedic implants.

One of the fundamental principles underlying tissue engineering approaches is that newly formed tissue must maintain sufficient vascularization to support its growth. Efforts to induce vascular growth into tissue-engineered scaffolds have recently been dedicated to developing novel strategies to deliver specific biological factors that direct the recruitment of endothelial cell (EC) progenitors and their differentiation. The challenge, however, lies in orchestration of the cells, appropriate biological factors, and optimal factor doses. This study reports an approach as a step forward to resolving this dilemma by combining an ex vivo gene transfer strategy and EC transplantation. The utility of this approach was evaluated by using 3D poly(lactide-co-glycolide) (PLAGA) sintered microsphere scaffolds for bone tissue engineering applications. Our goal was achieved by isolation and transfection of adipose-derived stromal cells (ADSCs) with adenovirus encoding the cDNA of VEGF. We demonstrated that the combination of VEGF releasing ADSCs and ECs results in marked vascular growth within PLAGA scaffolds. We thereby delineate the potential of ADSCs to promote vascular growth into biomaterials.

Achieving functional graft integration with subchondral bone poses a significant challenge for orthopaedic soft tissue repair and reconstruction. Soft tissues such as the anterior cruciate ligament (ACL) integrate with bone through a fibrocartilage interface, which minimizes stress concentrations and mediates load transfer between soft and hard tissues. We propose that biological fixation can be achieved by regenerating this fibrocartilage interface on biological or synthetic ACL grafts. This study focuses on the in vivo evaluation of a stratified scaffold predesigned to mimic the multitissue transition found at the ACL-to-bone interface. Specifically, the scaffold consists of three distinct yet continuous phases: Phase A for ligament formation, Phase B for the interface, and Phase C for the bone region. Interface-relevant cell types, specifically fibroblasts, chondrocytes, and osteoblasts, will be tri-cultured on this scaffold, and the formation of cell type- and phase-specific matrix heterogeneity as well as fibrocartilage formation will be evaluated over 8 weeks in a subcutaneous athymic rat model. Acellular scaffolds as well as scaffolds co-cultured with fibroblasts and osteoblasts will serve as controls. It was found that the triphasic scaffold supported multilineage cellular interactions as well as tissue infiltration and abundant matrix production in vivo. In addition, controlled phase-specific matrix heterogeneity was induced on the scaffold, with distinct mineral and fibrocartilage-like tissue regions formed in the tri-cultured group. Cell seeding had a positive effect on both host infiltration and matrix elaboration, which also translated into increased mechanical properties in the seeded groups compared to the acellular controls. In summary, the biomimetic and multiphasic design coupled with spatial control of cell distribution enables multitissue regeneration on the stratified scaffold, and demonstrates the potential for regenerating the interface between soft tissue grafts and bone., ((c) 2008 Wiley Periodicals, Inc.)

Soft tissues such as ligaments and tendons integrate with bone through a fibrocartilaginous interface divided into noncalcified and calcified regions. This junction between distinct tissue types is frequently injured and not reestablished after surgical repair. Its regeneration is also limited by a lack of understanding of the structure-function relationship inherent at this complex interface. Therefore, focusing on the insertion site between the anterior cruciate ligament (ACL) and bone, the objectives of this study are: (i) to determine interface compressive mechanical properties, (ii) to characterize interface mineral presence and distribution, and (iii) to evaluate insertion site-dependent changes in mechanical properties and matrix mineral content. Interface mechanical properties were determined by coupling microcompression with optimized digital image correlation analysis, whereas mineral presence and distribution were characterized by energy dispersive x-ray analysis and backscattered scanning electron microscopy. Both region- and insertion-dependent changes in mechanical properties were found, with the calcified interface region exhibiting significantly greater compressive mechanical properties than the noncalcified region. Mineral presence was only detectable within the calcified interface and bone regions, and its distribution corresponds to region-dependent mechanical inhomogeneity. Additionally, the compressive mechanical properties of the tibial insertion were greater than those of the femoral. The interface structure-function relationship elucidated in this study provides critical insight for interface regeneration and the formation of complex tissue systems.