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Particles released from implants cause inflammatory bone loss, which is a key factor in aseptic loosening, the most common reason for joint replacement failure. With the anticipated increased incidence of total joint replacement in the next decade, implant failure will continue to burden patients. The gut microbiome is increasingly recognized as an important factor in bone physiology, however, its role in implant loosening is currently unknown. We tested the hypothesis that implant loosening is associated with changes in the gut microbiota in a preclinical model. When the particle challenge caused local joint inflammation, decreased peri-implant bone volume, and decreased implant fixation, the gut microbiota was affected. When the particle challenge did not cause this triad of local effects, the gut microbiota was not affected. Our results suggest that cross-talk between these compartments is a previously unrecognized mechanism of failure following total joint replacement.

In 2021, the American Association for Anatomy (AAA) Board of Directors appointed a Task Force on Structural Racism to understand how the laws, rules, and practices in which the Association formed, developed and continues to exist affect membership and participation. This commentary is the first public report from the Task Force. We focus on African Americans with some comments on Jews and women, noting that all marginalized groups deserve study. Through much of its 130 year history, some members were an essential part of perpetuating racist ideas, the Association largely ignored racism and had some practices that prevented participation. The Task Force concluded that individual and structural racism within the AAA, combined with the broader social context in which the Association developed, contributed to the current underrepresentation of African Americans who constitute 4.1% of the membership even though 13.4% of the U.S. population is Black. Intentional efforts within the AAA to reckon with racism and other forms of bias have only begun in the last 10-20 years. These actions have led to more diverse leadership within the Association, and it is hoped that these changes will positively affect the recruitment and retention of marginalized people to science in general and anatomy in particular. The Task Force recommends that the AAA Board issue a statement of responsibility to acknowledge its history. Furthermore, the Task Force advocates that the Board commit to (a) sustaining ongoing projects to improve diversity, equity, and inclusion and (b) dedicating additional resources to facilitate novel initiatives.

Biomarkers are of interest to identify patients at risk for peri-implant osteolysis and aseptic loosening. We used a rat model of particle-induced peri-implant osteolysis to investigate if early changes in biomarkers were associated with subsequent implant fixation strength. Implants were placed in rat femora, which were then challenged with intra-articular knee injections of either clean polyethylene, lipopolysaccharide-doped polyethylene, or cobalt-chromium alloy particles, with particle-free vehicle serving as control (n ≥ 8 per group). Rats were weighed weekly, blood was collected at weeks 0, 3, 5, and 6, and locomotor behavior was assessed 4 days before study conclusion. Rats were euthanized 6 weeks post surgery. Week 6 serum was analyzed for five bone remodeling markers, while longitudinal serum was assessed for osteocalcin. Bone-implant contact, peri-implant trabecular architecture, and implant fixation strength were measured. Rats challenged with cobalt-chromium particles had a significant reduction in implant fixation strength compared with the vehicle-control group (P = .034). This group also had elevated serum osteocalcin (P = .005), depressed weight gain (P = .001) and less frequent rearing behavior (P = .029). Regardless of group, change in serum osteocalcin at week 3 (r = -.368; P = .046), change in weight at week 2 (r = .586; P < .001), as well as weight change at all other time intervals were associated with fixation strength. The finding that early alterations in serum osteocalcin and body weight were predictive of subsequent implant fixation strength supports continued investigation of biomarkers for early detection of peri-implant osteolysis and implant loosening. Further, change in biomarker levels was found to be more indicative of implant fixation status than any single measurement.

Mechanical fixation of the implant to host bone is an important contributor to orthopaedic implant survivorship. The relative importance of bone-implant contact, trabecular bone architecture, and cortical bone geometry to implant fixation strength has never been directly tested, especially in the settings of differential implant surface properties. Thus, using a rat model where titanium rods were placed into the intramedullary canal of the distal femur, we determined the relative contribution of bone-implant contact and peri-implant bone architecture to the fixation strength in implants with different surface roughness: highly polished and smooth (As-received) and dual acid etched (DAE) implants. Using a training set that maximized variance in implant fixation strength, we initially examined correlation between implant fixation strength and outcome parameters from microcomputed tomography and found that osseointegration volume per total volume (OV/TV), trabecular bone volume/total volume (BV/TV), and cortical thickness (Ct.Th) were the single best compartment-specific predictors of fixation strength. We defined separate regression models to predict implant fixation strength for As-received and DAE implants. When the training set models were applied to independent validation sets, we found strong correlations between predicted and experimentally measured implant fixation strength, with r(2) = 0.843 in As-received and r(2) = 0.825 in DAE implants. Interestingly, for As-received implants, OV/TV explained more of the total variance in implant fixation strength than the other variables, whereas in DAE implants, Ct.Th had the most explanatory power, suggesting that surface topography of implants affects which bone compartment is most important in providing implant fixation strength.

The circadian clock system regulates multiple metabolic processes, including bone metabolism. Previous studies have demonstrated that both central and peripheral circadian signaling regulate skeletal growth and homeostasis. Disruption in central circadian rhythms has been associated with a decline in bone mineral density and the global and osteoblast-specific disruption of clock genes in bone tissue leads to lower bone mass. Gut physiology is highly sensitive to circadian disruption. Since the gut is also known to affect bone remodeling, we sought to test the hypothesis that circadian signaling disruption in colon epithelial cells affects bone. We therefore assessed structural, functional, and cellular properties of bone in 8 week old Ts4-Cre and Ts4-Cre;Bmal1fl/fl (cBmalKO) mice, where the clock gene Bmal1 is deleted in colon epithelial cells. Axial and appendicular trabecular bone volume was significantly lower in cBmalKO compared to Ts4-Cre 8-week old mice in a sex-dependent fashion, with male but not female mice showing the phenotype. Similarly, the whole bone mechanical properties were deteriorated in cBmalKO male mice. The tissue level mechanisms involved suppressed bone formation with normal resorption, as evidenced by serum markers and dynamic histomorphometry. Our studies demonstrate that colon epithelial cell-specific deletion of Bmal1 leads to trabecular and cortical bone loss in male mice.

To assess in-vitro trabecular bone damage following mono- and bicortical mini-implant (MI) anchorage in mini-implant assisted rapid palatal expansion (MARPE).Sixteen self-drilling MI (four MARPE appliances) were distributed in two groups according to bone insertion (monocortical and bicortical) in bovine rib. The device was activated five times (0.5mm each). Trabecular bone damage was assessed using micro-CT scans made at baseline and after each activation by trabecular spacing parameter (Tb.Sp) (distance [mm] between the trabecular bone structure). These measurements were made in five different regions of interest (ROI) surrounding the screw (whole, superior, inferior, anterior and posterior). Two-way ANOVA with Tukey post-hoc analysis (α=0.05) was used to evaluate the effect of insertion type (monocortical vs. bicortical) and activation cycle (0-5) on trabecular damage. The time effect was evaluated using ANOVA-MR test effect with Bonferroni correction (α=0.003). The micro-CT images were also examined qualitatively.When analysing the individual ROIs, only the superior ROI had a significant difference (P0.003) beginning at the fourth activation cycle. For the monocortical group, trabecular spacing was affected when the whole ROI was analysed beginning at the fourth activation cycle, while for the superior ROI, this difference became apparent beginning with the third activation cycle (P0.003). For the qualitative analysis, it seems that only monocortical anchorage influences the trabecular bone in the superior area.Monocortical anchorage is more susceptible to bone damage around the MIs, with the superior (cervical) region most strongly affected.

Background Matched serum and urine samples from patients who had total hip replacement were used to assess serum-validated immunoassay reagents for use in urine. Methods Samples were evaluated by an automated electrochemiluminescent immunoassay (cobas e411; Roche Diagnostics) for C-terminal telopeptide of type I collagen isoform β (β-Crosslaps), osteocalcin N-terminal midfragment (N-MID OC), N-terminal propeptide of type I collagen (PINP), and interleukin 6 (IL-6). Spike and recovery experiments were utilized to assess urinary matrix effects. Correlations between serum and both raw and creatinine-corrected urinary measures were assessed. Short-term precision was assessed. Results Spike and recovery experiments indicated minimal matrix effects of urine for the β-Crosslaps assay. Potential matrix effects were observed for the other analytes because N-MID OC and IL-6 tended to be slightly overrecovered, whereas PINP was underrecovered. There were strong correlations between serum β-Crosslaps and raw (Spearman ρ [rs] = 0.725, P < 0.0001) and creatinine-corrected (rs = 0.793, P < 0.0001) urinary measures and moderate correlations between serum N-MID OC and raw (rs = 0.582, P < 0.0001) and creatinine-corrected (rs = 0.482, P < 0.0001) urinary measures. PINP was not detected in urine, and no significant serum–urine correlations were found for IL-6. Short-term precision for urinary levels of β-Crosslaps, N-MID OC, and IL-6 were 1.6%, 6.3% and 14.4%, respectively. Conclusions Urinary measurements of β-Crosslaps and N-MID OC assays were correlated with serum measurements and had good short-term precision. Urinary PINP was not detectable. IL-6 can be measured in urine using this technology, but the levels did not correlate with serum levels, and the short-term precision was variable.

Peri-implant osteolysis is commonly diagnosed after substantial bone loss has occurred, making revision surgery more challenging. The goal of the current study was to identify urinary biomarkers that differentiate total hip replacement patients who eventually develop osteolysis from patients who do not. We used a repository of 24-h urine samples collected prior to surgery and annually thereafter in 26 patients, 16 who developed osteolysis, and 10 who did not. We examined the markers at radiographic diagnosis, annually for 6 years preceding diagnosis, at the first post-operative sampling point, and pre-operatively. Patients in the osteolysis and non-osteolysis groups were matched according to time post-surgery and did not differ in the male:female ratio or age at surgery. Seven candidate biomarkers were measured, including free deoxypyridinoline (DPD), cross-linked N-telopeptides (NTX), interleukin-6 (IL-6), interleukin-8 (IL-8), osteoprotegerin (OPG), α-crosslaps (α-CTX), and β-crosslaps (β-CTX). As an individual biomarker, DPD demonstrated the highest ability to predict osteolysis, with an area under the curve (AUC) in Receiver Operating Characteristic (ROC) analyses of 0.844 at 6 years prior to diagnosis. A panel of α-CTX and IL-6 was able to identify at-risk patients with an AUC of 0.941 or greater at all post-operative time points and an AUC of 1.000 pre-operatively. The results demonstrate the potential of using non-invasive biomarkers to identify patients at risk for peri-implant osteolysis long before the emergence of radiographic signs. Further, the high accuracy of the pre-operative biomarker levels demonstrates the potential importance of pre-existing, patient-specific factors driving subsequent osteolysis. Study Design © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2754-2761, 2018.

BACKGROUND: Osteoporosis and Alzheimer’s disease are common diseases of aging that would seem to be unrelated, but may be linked through the influence of bone-derived signals on brain function. The aim of the current study is to investigate the relationship between circulating levels of bone-related biomarkers and cognition. METHODS: The population included 103 community-dwelling older individuals with memory concerns but without cognitive impairment. A global cognition summary measure was collected at baseline and 6, 12, and 18 months post-enrollment by converting raw scores from 19 cognitive function tests to z-scores and averaging. Baseline plasma concentrations of bone-related biomarkers, including undercarboxylated, carboxylated, and total osteocalcin, parathyroid hormone, C-terminal telopeptide of collagen 1 (CTX-1), procollagen type 1 amino-terminal propeptide, osteoprotegrin, osteopontin, Dickkopf WNT signaling pathway inhibitor 1 (Dkk1), sclerostin, and amyloid β peptides (Aβ(40) and Aβ(42)), were measured. RESULTS: Using sex, age, and education-adjusted mixed-effects models, we found that baseline levels of TNF-related apoptosis-inducing ligand (TRAIL; p < .001), Dkk1 (p = .014), and CTX-1 (p = .046) were related to the annual rate of change of global cognition over the 18 month follow-up. In cognitive domain-specific analysis, baseline TRAIL was found to be positively related to the annual rate of change in episodic (p < .001) and working memory (p = .016), and baseline Dkk1 was positively related to semantic memory (p = .027) and negatively related to working memory (p = .016). CONCLUSIONS: These results further confirm the link between bone and brain health and suggest that circulating levels of bone-related biomarkers may have diagnostic potential to predict worsening cognition.

We completed a systematic literature review of in vivo animal models that use arthrotomy-based methods to study particle-induced peri-implant osteolysis. The purpose of the review was to characterize the models developed to date, to determine the questions addressed, to assess scientific rigor and transparency, and to identify gaps in knowledge. We probed three literature databases (Medline, Embase, and Scopus) and found 77 manuscripts that fit the search parameters. In the most recent 10 years, researchers mainly used rat and mouse models, whereas in the previous 20 years, large animal, canine, and rabbit models were more common. The studies have demonstrated several pathophysiology pathways, including macrophage migration, particle phagocytosis, increased local production of cytokines and lysosomal enzymes, elevated bone resorption, and suppressed bone formation. The effect of variation in particle characteristics and concentration received limited attention with somewhat mixed findings. Particle contamination by endotoxin was shown to exacerbate peri-implant osteolysis. The possibility of early diagnosis was demonstrated through imaging and biomarker approaches. Several studies showed that both local and systemic delivery of bisphosphonates inhibits the development of particle-induced osteolysis. Other methods of inhibiting osteolysis include the use of anabolic agents and altering the implant design. Few studies examined non-surgical rescue of loosened implants, with conflicting results with alendronate. We found that the manuscripts often lacked the methodological detail now advocated by the ARRIVE guidelines, suggesting that improvement in reporting would be useful to maximize rigor and transparency. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2595-2605, 2017.

Articular cartilage lines the load-bearing surfaces of long bones and undergoes compositional and structural degeneration during osteoarthritis progression. Contrast enhanced microcomputed tomography (μCT) is being applied to a variety of preclinical models, including the mouse, to map structural and compositional properties in 3-D. The thinness (˜30-50 μm) and high cellularity of mouse articular cartilage presents a significant imaging challenge. Our group previously showed that mouse articular cartilage and proteoglycan (PG) content can be assessed by μCT with the ioxagalate-based contrast agent Hexabrix, but the voxel size used (6 μm) was deemed to be barely adequate. The objective of the present study is to assess the utility of a novel contrast agent, CA4+, to quantify mouse articular cartilage morphology and composition with high resolution μCT imaging (3 μm voxels) and to compare the sensitivity of CA4+ and Hexabrix to detect between-group differences. While both contrast agents are iodine-based, Hexabrix is anionic and CA4+ is cationic so they interact differently with negatively charged PGs. With CA4+, a strong correlation was found between non-calcified articular cartilage thickness measurements made with histology and μCT (R2 = 0.72, p < 0.001). Cartilage degeneration - as assessed by loss in volume, thickness and PG content - was observed in 34-week old mice when compared to both 7- and 12-week old mice. High measurement precision was observed with CA4+, with the coefficient of variation after repositioning and re-imaging samples equaling 2.8%, 4.5%, 7.4% and 5.9% for attenuation, thickness, volume, and PG content, respectively. Use of CA4+ allowed increased sensitivity for assessing PG content compared to Hexabrix, but had no advantage for measurement of cartilage thickness or volume. This improvement in imaging should prove useful in preclinical studies of cartilage degeneration and regeneration. This article is protected by copyright. All rights reserved

Various intact and post-injury bone phenotypes are heritable traits. In this study, we sought to determine if intramembranous bone regeneration following marrow ablation differed among common inbred mouse strains and to identify how early the differences appear. We found a ∼four-fold difference in the regenerated bone volume 21 days after marrow ablation in females from four inbred mouse strains: FVB/N (15.7 ± 8.1%, mean and standard deviation), C3H/He (15.5 ± 4.2%), C57BL/6 (12.2 ± 5.2%), and BALB/c (4.0 ± 4.4%); with BALB/c different from FVB/N (p = 0.007) and C3H/He (p = 0.002). A second experiment showed that FVB/N compared to BALB/c mice had more regenerated bone 7 and 14 days after ablation (p

Background: The mechanical fixation of orthopaedic and dental implants is compromised by diminished bone volume, such as with osteoporosis. Systemic administration of sclerostin antibody (Scl-Ab) has been shown to enhance implant fixation in normal animals. In the present study, we tested whether Scl-Ab can improve implant fixation in established osteoporosis in a rat model. Methods: We used an ovariectomized (ovx) rat model, in which we found a 78% decrease in trabecular bone volume at the time of implant surgery; sham-ovx, age-matched rats were used as controls. After placement of a titanium implant in the medullary cavity of the distal aspect of the femur, the rats were maintained for four, eight, or twelve weeks and treated biweekly with Scl-Ab or with the delivery vehicle alone. Outcomes were measured with use of microcomputed tomography, mechanical testing, and static and dynamic histomorphometry. Results: Scl-Ab treatment doubled implant fixation strength in both the sham-ovx and ovx groups, although the enhancement was delayed in the ovx group. Scl-Ab treatment also enhanced bone-implant contact; increased peri-implant trabecular thickness and volume; and increased cortical thickness. These structural changes were associated with an approximately five to sevenfold increase in the bone-formation rate and a >50% depression in the eroded surface following Scl-Ab treatment. Trabecular bone thickness and bone-implant contact accounted for two-thirds of the variance in fixation strength. Conclusions: In this model of severe osteoporosis, Scl-Ab treatment enhanced implant fixation by stimulating bone formation and suppressing bone resorption, leading to enhanced bone-implant contact and improved trabecular bone volume and architecture. Clinical Relevance: Systemic administration of anti-sclerostin antibodies might be a useful strategy in total joint replacement when bone mass is deficient.

Histology and backscatter scanning electron microscopy (bSEM) are the current gold standard methods for quantifying bone-implant contact (BIC), but are inherently destructive. Microcomputed tomography (μCT) is a non-destructive alternative, but attempts to validate μCT-based assessment of BIC in animal models have produced conflicting results. We previously showed in a rat model using a 1.5 mm diameter titanium implant that the extent of the metal-induced artefact precluded accurate measurement of bone sufficiently close to the interface to assess BIC. Recently introduced commercial laboratory μCT scanners have smaller voxels and improved imaging capabilities, possibly overcoming this limitation. The goals of the present study were to establish an approach for optimizing μCT imaging parameters and to validate μCT-based assessment of BIC. In an empirical parametric study using a 1.5 mm diameter titanium implant, we determined 90 kVp, 88 µA, 1.5 μm isotropic voxel size, 1600 projections/180°, and 750 ms integration time to be optimal. Using specimens from an in vivo rat experiment, we found significant correlations between bSEM and μCT for BIC with the manufacturer's automated analysis routine (r = 0.716, p = 0.003) or a line-intercept method (r = 0.797, p = 0.010). Thus, this newer generation scanner's improved imaging capability reduced the extent of the metal-induced artefact zone enough to permit assessment of BIC. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 9 999:XX–XX, 2017.

Particle-induced osteolysis is caused by an imbalance in bone resorption and formation, often leading to loss of implant fixation. Bone remodeling biomarkers may be useful for identification of osteolysis and studying pathogenesis, but interpretation of biomarker data could be confounded if local osteolysis engenders systemic bone remodeling. Our goal was to determine if remote bone remodeling contributes to biomarker levels. Serum concentrations of eight biomarkers and bone remodeling rates at local (femur), contiguous (tibia), and remote (humerus and lumbar vertebra) sites were evaluated in a rat model of particle-induced osteolysis. Serum CTX-1, cathepsin K, PINP, and OPG were elevated and osteocalcin was suppressed in the osteolytic group, but RANKL, TRAP 5b, and sclerostin were not affected at the termination of the study at 12 weeks. The one marker tested longitudinally (CTX-1) was elevated by 3 weeks. We found increased bone resorption and decreased bone formation locally, subtle differences in contiguous sites, but no differences remotely at 12 weeks. Thus, the skeletal response to local particle challenge was not systemic, implying that the observed differences in serum biomarker levels reflect differences in local remodeling.

Osteoporosis presents a challenge for successful implant fixation due to an impaired healing response. Preclinical studies have consistently reported reduced osseointegration capability in trabecular bone. Although clinical studies of implant success in dentistry have not found a negative effect due to osteoporosis, low bone mass is a significant risk factor for implant migration in orthopedics. Pharmacologic treatment options that limit bone resorption or upregulate formation have been studied preclinically. While, both treatment options improve implant fixation, direct comparisons to-date have found anti-catabolic more effective than anabolic treatments for establishing implant fixation, but combination approaches are better than either treatment alone. Clinically, anti-catabolic treatments, particularly bisphosphonates have been shown to increase the longevity of implants, while limited clinical evidence on the effects of anabolic treatment exists. Preclinical experiments are needed to determine the effects of osteoporosis and subsequent treatment on the long-term maintenance of fixation and recovery after bone loss.

Articular cartilage lines the load-bearing surfaces of long bones and undergoes compositional and structural degeneration during osteoarthritis progression. Contrast enhanced microcomputed tomography (μCT) is being applied to a variety of preclinical models, including the mouse, to map structural and compositional properties in 3-D. The thinness (~30–50 μm) and high cellularity of mouse articular cartilage presents a significant imaging challenge. Our group previously showed that mouse articular cartilage and proteoglycan (PG) content can be assessed by μCT with the ioxagalate-based contrast agent Hexabrix, but the voxel size used (6 μm) was deemed to be barely adequate. The objective of the present study is to assess the utility of a novel contrast agent, CA4+, to quantify mouse articular cartilage morphology and composition with high resolution μCT imaging (3 μm voxels) and to compare the sensitivity of CA4+ and Hexabrix to detect between-group differences. While both contrast agents are iodine-based, Hexabrix is anionic and CA4+ is cationic so they interact differently with negatively charged PGs. With CA4+, a strong correlation was found between non-calcified articular cartilage thickness measurements made with histology and μCT (R2 = 0.72, p < 0.001). Cartilage degeneration – as assessed by loss in volume, thickness and PG content – was observed in 34-week old mice when compared to both 7- and 12-week old mice. High measurement precision was observed with CA4+, with the coefficient of variation after repositioning and re-imaging samples equaling 2.8%, 4.5%, 7.4% and 5.9% for attenuation, thickness, volume, and PG content, respectively. Use of CA4+ allowed increased sensitivity for assessing PG content compared to Hexabrix, but had no advantage for measurement of cartilage thickness or volume. This improvement in imaging should prove useful in preclinical studies of cartilage degeneration and regeneration.

In this paper, we provide a detailed protocol for a model of long bone mechanical marrow ablation in the rodent, including surgical procedure, anesthesia, and pre- and post-operative care. In addition, frequently used experimental end points are briefly discussed. This model was developed to study intramembranous bone regeneration following surgical disruption of the marrow contents of long bones. In this model, the timing of the appearance of bone formation and remodeling is well-characterized and therefore the model is well-suited to evaluate the in vivo effects of various agents which influence these processes. When biomaterials such as tissue engineering scaffolds or metal implants are placed in the medullary cavity after marrow ablation, end points relevant to tissue engineering and implant fixation can also be analyzed. By sharing a detailed protocol, we hope to improve inter-laboratory reproducibility.

To examine bone remodeling following implant placement, 88 female Sprague–Dawley rats underwent either sham ovariectomy (sham-ovx) or ovariectomy (ovx) at 4.5 months. At 11 months, 17 baseline control animals were euthanized, while 71 rats received bilateral intramedullary femoral implants. Implanted rats were randomized to 4-, 8-, or 12-week follow-up times. Microcomputed tomography was used to assess cortical area and trabecular architecture in all rats. Dynamic and static histomorphometry were performed in a subset to examine the trabecular and endocortical bone in the distal femoral metaphysis adjacent to the implant and the periosteal surface at the midshaft superior to the implant (n = 59). Implantation did not affect bone volume in either sham-ovx or ovx rats compared to baseline controls. Implant placement significantly increased mineralizing surface, mineral apposition rate, and bone formation rate in both sham-ovx and ovx rats at the trabecular and endocortical surfaces at four and sometimes 8 weeks, with a return to baseline values by 12 weeks. At the periosteal surface, implant placement increased bone formation at 4 weeks with a return to baseline levels by 8 weeks. Thus, implant placement increases bone remodeling transiently without affecting bone volume in sham-ovx and ovx rats. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 800–806, 2013

Objective To assess the ability of sclerostin antibody therapy to blunt the negative effects of polyethylene particles on implant fixation and peri-implant bone structure in a rat implant fixation model. Methods Thirty-six adult male rats received intramedullary titanium implants; 12 rats received vehicle injections only (control), and 24 rats received intraarticular injections of lipopolysaccharide-doped polyethylene particles. Twelve of the rats that received particles also received sclerostin antibody treatment. The 3 groups of rats were maintained for 12 weeks in a pathogen-free environment, at which time mechanical, micro–computed tomography, and dynamic and static histomorphometry end points were assessed. Results Sclerostin antibody treatment completely blocked the negative effect of the lipopolysaccharide-doped polyethylene particles on implant fixation and peri-implant bone volume by increasing the bone formation rate and depressing bone resorption. Conclusion Anabolic agents targeting the Wnt signaling pathway are a promising new alternative for the prevention of periprosthetic osteolysis and aseptic loosening.

Background: Previous studies have demonstrated that sclerostin blockade is anabolic for bone. This study examined whether systemic administration of sclerostin antibody would increase implant fixation and peri-implant bone volume in a rat model. Methods: Titanium cylinders were placed in the femoral medullary canal of ninety male Sprague-Dawley rats. One-half of the rats (n = 45) received murine sclerostin antibody (Scl-Ab, 25 mg/kg, twice weekly) and the other one-half (n = 45) received saline solution. Equal numbers of rats from both groups were sacrificed at two, four, or eight weeks after the implant surgery and the femora were examined by microcomputed tomography, mechanical pull-out testing, and histology. Results: Fixation strength in the two groups was similar at two weeks but was 1.9-fold greater at four weeks (p = 0.024) and 2.2-fold greater at eight weeks (p < 0.001) in the rats treated with sclerostin antibody. At two weeks, antibody treatment led to increased cortical area, with later increases in cortical thickness and total cross-sectional area. Significant differences in peri-implant trabecular bone were not evident until eight weeks but included increased bone volume per total volume, bone structure that was more plate-like, and increased trabecular thickness and number. Changes in bone architecture in the intact contralateral femur tended to precede the peri-implant changes. The peri-implant bone properties accounted for 61% of the variance in implant fixation strength, 32% of the variance in stiffness, and 63% of the variance in energy to failure. The implant fixation strength at four weeks was approximately equivalent to the strength in the control group at eight weeks. Conclusions: Sclerostin antibody treatment accelerated and enhanced mechanical fixation of medullary implants in a rat model by increasing both cortical and trabecular bone volume. Clinical Relevance: Sclerostin antibody treatment may be useful for improving implant fixation.

Enhancement of in vivo mobilization and homing of endogenous mesenchymal stem cells (MSCs) to an injury site is an innovative strategy for improvement of bone tissue engineering and repair. The present study was designed to determine whether mobilization by AMD3100 and/or local homing by delivery of stromal cell-derived factor-1 (SDF-1) enhances recombinant human bone morphogenetic protein-2 (rhBMP-2) induced ectopic bone formation in an established rat model. Rats received an injection of either saline or AMD3100 treatment 1 h before harvesting of bone marrow for in vitro colony-forming unit-fibroblasts (CFU-F) culture or the in vivo subcutaneous implantation of absorbable collagen sponges (ACSs) loaded with saline, recombinant human bone morphogenetic protein-2 (rhBMP-2), SDF-1, or the combination of SDF-1 and rhBMP-2. AMD3100 treatment resulted in a significant decrease in CFU-F number, compared with saline, which confirmed that a single systemic AMD3100 treatment rapidly mobilized MSCs from the bone marrow. At 28 and 56 days, bone formation in the explanted ACS was assessed by microcomputed tomography (μCT) and histology. At 28 days, AMD3100 and/or SDF-1 had no statistically significant effect on bone volume (BV) or bone mineral content (BMC), but histology revealed more active bone formation with treatment of AMD3100, loading of SDF-1, or the combination of both AMD3100 and SDF-1, compared with saline-treated rhBMP-2 loaded ACS. At 56 days, the addition of AMD3100 treatment, loading of SDF-1, or the combination of both resulted in a statistically significant stimulatory effect on BV and BMC, compared with the saline-treated rhBMP-2 loaded ACS. Histology of the 56-day ACS were consistent with the μCT analysis, exhibiting more mature and mineralized bone formation with AMD3100 treatment, SDF-1 loading, or the combination of both, compared with the saline-treated rhBMP-2 loaded ACS. The present study is the first that provides evidence of the efficacy of AMD3100 and SDF-1 treatment to stimulate trafficking of MSCs to an ectopic implant site, in order to ultimately enhance rhBMP-2 induced long-term bone formation.

Revision surgery for particle-induced implant loosening in total joint replacement is expected to increase dramatically over the next few decades. This study was designed to investigate if local tissue and serum markers of bone remodeling reflect implant fixation following administration of lipopolysaccharide (LPS)-doped polyethylene (PE) particles in a rat model. 24 rats received bilateral implantation of intramedullary titanium rods in the distal femur, followed by weekly bilateral intra-articular injection of either LPS-doped PE particles (n = 12) or vehicle which contained no particles (n= 12) for 12 weeks. The group in which the particles were injected had increased serum C-terminal telopeptide of type I collagen, decreased serum osteocalcin, increased peri-implant eroded surface, decreased peri-implant bone volume, and decreased mechanical pull-out strength compared to the controls. Implant fixation strength was positively correlated with peri-implant bone volume and serum osteocalcin and inversely correlated with serum C-terminal telopeptide of type I collagen, while energy to yield was positively correlated with serum osteocalcin and inversely correlated with the number of tartrate resistant acid phosphatase positive cells at the interface and the amount of peri-implant eroded surface. There was no effect on trabecular bone volume at a remote site. Thus, the particle-induced impaired fixation in this rat model was directly associated with local and serum markers of elevated bone resorption and depressed bone formation, supporting the rationale of exploring both anti-catabolic and anabolic strategies to treat and prevent particle-related implant osteolysis and loosening and indicating that serum markers may prove useful in tracking implant fixation.

Fixation of metallic implants to bone through osseointegration is important in orthopaedics and dentistry. Model systems for studying this phenomenon would benefit from a non-destructive imaging modality so that mechanical and morphological endpoints can more readily be examined in the same specimens. The purpose of this study was to assess the utility of an automated microcomputed tomography (μCT) program for predicting bone-implant contact (BIC) and mechanical fixation strength in a rat model. Femurs in which 1.5-mm-diameter titanium implants had been in place for 4 weeks were either embedded in polymethylmethacrylate (PMMA) for preparation of 1-mm-thick cross-sectional slabs (16 femurs: 32 slabs) or were used for mechanical implant pull-out testing (n= 18 femurs). All samples were scanned by μCT at 70 kVp with 16 μm voxels and assessed by the manufacturer's software for assessing 'osseointegration volume per total volume' (OV/TV). OV/TV measures bone volume per total volume (BV/TV) in a 3-voxel-thick ring that by default excludes the 3 voxels immediately adjacent to the implant to avoid metal-induced artefacts. The plastic-embedded samples were also analysed by backscatter scanning electron microscopy (bSEM) to provide a direct comparison of OV/TV with a well-accepted technique for BIC. In μCT images in which the implant was directly embedded within PMMA, there was a zone of elevated attenuation (>50% of the attenuation value used to segment bone from marrow) which extended 48 μm away from the implant surface. Comparison of the bSEM and μCT images showed high correlations for BV/TV measurements in areas not affected by metal-induced artefacts. In addition for bSEM images, we found that there were high correlations between peri-implant BV/TV within 12 μm of the implant surface and BIC (correlation coefficients ≥0.8, p < 0.05). OV/TV as measured on μCT images was not significantly correlated with BIC as measured on the corresponding bSEM images. However, OV/TV was significantly, but weakly, correlated with implant pull-out strength (r= 0.401, p= 0.049) and energy to failure (r= 0.435, p= 0.035). Thus, the need for the 48-μm-thick exclusion zone in the OV/TV program to avoid metal-induced artefacts with the scanner used in this study means that it is not possible to make bone measurements sufficiently close to the implant surface to obtain an accurate assessment of BIC. Current generation laboratory-based μCT scanners typically have voxel sizes of 6-8 μm or larger which will still not overcome this limitation. Thus, peri-implant bone measurements at these resolutions should only be used as a guide to predict implant fixation and should not be over-interpreted as a measurement of BIC. Newer generation laboratory-based μCT scanners have several improvements including better spatial resolution and X-ray sources and appear to have less severe metal-induced artefacts, but will need appropriate validation as they become available to researchers. Regardless of the μCT scanner being used, we recommend that detailed validation studies be performed for any study using metal implants because variation in the composition and geometry of the particular implants used may lead to different artefact patterns.

Aseptic loosening is the devastating long term complication of total hip arthroplasty and orthopedic implant debris has been shown to trigger an intense inflammatory reaction leading to resorption of the bone matrix. Inflammatory cytokines, such as tumor necrosis factor-α (TNFα), have been implicated in this process and osteocytes may play a role in its production. We previously demonstrated that cobalt-chromium-molybdenum (CoCrMo) particles upregulate TNFα production by MLO-Y4 osteocytes in vitro, but the underlying mechanism has not been elucidated. Based on previous studies by others, we hypothesized that the calcineurin-nuclear factor of activated T cells (NFAT) pathway mediates CoCrMo particle-induced TNFα production in MLO-Y4 osteocytes. MLO-Y4 osteocytes exposed to CoCrMo particle treatment resulted in a rapid and significant increase in calcineurin activity. We also demonstrate that CoCrMo particle-induced upregulation of TNFα is reduced to control levels with calcineurin-NFAT inhibitors and this was also confirmed at mRNA level. Moreover, we demonstrate the localization of NFATs in MLO-Y4 osteocytes and that NFAT1 and 2 translocate to the nucleus upon CoCrMo particle treatment. Our results suggest that calcineurin-NFAT signaling is involved in TNFα production by MLO-Y4 osteocytes after CoCrMo particle treatment.

Bone growth and repair are under the control of biochemical and mechanical signals. Low-intensity pulsed ultrasound (LIPUS) stimulation at 30mW/cm(2) is an established, widely used and FDA approved intervention for accelerating bone healing in fractures and non-unions. Although this LIPUS signal accelerates mineralization and bone regeneration, the actual intensity experienced by the cells at the target site might be lower, due to the possible attenuation caused by the overlying soft tissue. The aim of this study was to investigate whether LIPUS intensities below 30mW/cm(2) are able to provoke phenotypic responses in bone cells. Rat bone marrow stromal cells were cultured under defined conditions and the effect of 2, 15, 30mW/cm(2) and sham treatments were studied at early (cell activation), middle (differentiation into osteogenic cells) and late (biological mineralization) stages of osteogenic differentiation. We observed that not only 30mW/cm(2) but also 2 and 15mW/cm(2), modulated ERK1/2 and p38 intracellular signaling pathways as compared to the sham treatment. After 5 days with daily treatments of 2, 15 and 30mW/cm(2), alkaline phosphatase activity, an early indicator of osteoblast differentiation, increased by 79%, 147% and 209%, respectively, compared to sham, indicating that various intensities of LIPUS were able to initiate osteogenic differentiation. While all LIPUS treatments showed higher mineralization, interestingly, the highest increase of 225% was observed in cells treated with 2mW/cm(2). As the intensity increased to 15 and 30mW/cm(2), the increase in the level of mineralization dropped to 120% and 82%. Our data show that LIPUS intensities lower than the current clinical standard have a positive effect on osteogenic differentiation of rat bone marrow stromal cells. Although Exogen™ at 30mW/cm(2) continues to be effective and should be used as a clinical therapy for fracture healing, if confirmed in vivo, the increased mineralization at lower intensities might be the first step towards redefining the most effective LIPUS intensity for clinical use.

The presence of asymmetry in tibial bone mineral content (BMC) of the operated and control limbs at the end of the experimental period following unilateral hip replacement surgery is used as a marker of limb function. The goal of the present study was to determine the contribution of ipsilateral and contralateral bone gain and loss to control-treated side differences in BMC of the tibia in dogs following unilateral hip replacement surgery. Seven animals were followed longitudinally with single beam photon absorptiometry for 6 months after unilateral hip hemiarthroplasty. Bone loss, compared with preoperative baseline values, was observed in both limbs, with recovery in bone mass beginning 1 month after surgery in the contralateral tibia and 3 months after surgery in the ipsilateral tibia. Thus, the asymmetry in tibial BMC frequently seen after unilateral experimental hip replacement in the canine appears to be caused by differential timing of recovery of bone mass following a transient loss in both limbs. The mechanism defined in this study is in contrast to an alternative mechanism involving bone loss in the treated limb coupled with bone gain in the control limb.

Two methods to improve bone repair include the use of recombinant human bone morphogenetic protein-2 (rhBMP-2) and low-intensity pulsed ultrasound (LIPUS). The present study was designed to determine if LIPUS enhances the effect of rhBMP-2–induced bone formation in a well characterized ectopic implant model. Absorbable collagen sponges loaded with 0-, 1-, 2.5- or 5-μg doses of rhBMP-2 were implanted subcutaneously in 11-week-old, male Long Evans rats, followed by daily 20-min LIPUS or sham LIPUS treatment beginning 1 d after surgery. Explanted sponges were assessed for bone volume, mineral density and mineral content by microcomputed tomography (μCT). At two weeks, LIPUS had no effect on rhBMP-2–induced bone formation, but at four weeks, LIPUS increased bone volume in the 1-μg rhBMP-2–treated implants 117.7-fold (0.02 ± 0.04 mm 3 vs. 2.07 S.E.M. ± 1.67 mm 3 ; p = 0.028), and 2.3-fold in the 5-μg dose implants (5.96 ± 3.68 mm 3 vs. 13.52 ± 6.81 mm 3 ; p = 0.077) compared with sham LIPUS. Bone mineral density was not affected by LIPUS treatment. Total mineral content followed the same pattern as bone volume. Histologic staining for mineralized tissue was consistent with the μCT observations. The present study is the first to demonstrate that LIPUS enhances bone formation induced by rhBMP-2. (E-mail: robert_leven@rush.edu)

Limb bones deform during locomotion and can resist the deformations by adjusting their shapes. For example, a tubular-shaped diaphysis best resists variably-oriented deformations. As behavioral profiles change during adulthood, patterns of bone deformation may exhibit age trends. Habitat characteristics, e.g., annual rainfall, tree density, and elevation changes, may influence bone deformations by eliciting individual components of behavioral repertoires and suppressing others, or by influencing movements during particular components. Habituated chimpanzee communities provide a unique opportunity to examine these factors because of the availability of morphological data and behavioral observations from known-age individuals inhabiting natural habitats. We evaluated adult femora and humeri of 18 female and 10 male free-ranging chimpanzees (Pan troglodytes) from communities in Gombe (Tanzania), Mahale Mountains (Tanzania), and Tai Forest (Cote d’Ivoire) National Parks. We compare cross sections at several locations (35%, 50%, 65% diaphyseal lengths). Community comparisons highlight different diaphyseal shapes of Tai females relative to Mahale and Gombe females, particularly in humeral diaphyses. Age trends in diaphyseal shapes are consistent with reduced activity levels in general, not only reduced arboreal activity. Age-related bone loss is apparent among community females, but is less striking among males. Community trends in diaphyseal shape are qualitatively consistent with ranked annual rainfall at localities, tree density, and elevation change or ruggedness of terrain. Habitat characteristics may contribute to variation in diaphyseal shape among chimpanzee communities, much like among modern human groups, but verification awaits further rigorous experimental and comparative analyses.

Short-term osseointegration of orthopedic implants is critical for the long-term stability of the implant–bone interface. To improve initial implant stability, one strategy under consideration involves the presentation of adhesion ligands on the implant surface to stimulate bone regeneration in the peri-implant region. To assess the relative effects of implant surface chemistry and topography on osseointegration within the rat femoral ablation implant model, a nonfouling, enzymatically degradable interpenetrating polymer network (edIPN) of poly(AAm-co-EG/AAc) amenable to presenting the cell signaling domain Arg-Gly-Asp (RGD), was developed. Moderate enhancement of peri-implant bone formation was found after 28 days using the edIPN without peptide modification (p = 0.032). However, no data supported a benefit of peptide modification, as bone–implant contact, normalized bone volume and normalized fixation strength was equivalent or poorer than dual acid-etched (DAE) treated implants after 28 days. Surface topography was determined to be the dominant factor in modulating osseointegration, as DAE implants produced equivalent roughness-normalized fixation strength versus previously reported data on plasma-sprayed hydroxyapatite/tricalcium phosphate-coated implants (Barber et al., J Biomed Mater Res A, forthcoming). An ideal osseointegrated implant will require optimization of all three aforementioned parameters, and may take the form of biomolecule delivery from thin degradable polymer networks. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2007

Objective The relationship between knee pain and radiographic evidence of knee osteoarthritis (OA) is notoriously imperfect. In particular, conditions that distinguish individuals with symptoms from those with comparable radiographic involvement who remain asymptomatic are unclear. We investigated dynamic loading across the knee in individuals with mild radiographic OA who were distinguished by the presence or absence of knee pain. Methods Subjects were recruited into 3 groups: symptomatic with a Kellgren/Lawrence (K/L) grade of 2 (n = 52), asymptomatic with a K/L grade of 2 (n = 19), and asymptomatic with a K/L grade of 0 or 1 (n = 37), the latter representing a normal comparator group. Dynamic knee loading was assessed with gait analysis, and both the peak external knee adduction moment and the knee adduction angular impulse were determined. Results Peak knee adduction moment and knee adduction angular impulse were 19% and 30% higher, respectively, in symptomatic K/L grade 2 individuals than in asymptomatic individuals with the same radiographic grade (P < 0.05). Conversely, the asymptomatic K/L grade 2 group did not differ from the K/L grade 0–1 normal comparator group (P = 1.00). Conclusion Among individuals with mild radiographic knee OA (K/L grade 2), those who are symptomatic have significantly higher medial compartment loads than those who are asymptomatic, whereas those who are asymptomatic do not differ from normal controls (asymptomatic K/L grade 0 or 1). These findings suggest a biomechanical component to the distinction between asymptomatic and symptomatic radiographic OA, which may be pathophysiologically important.

We demonstrate that a biomimetic polymer network is capable of affecting bone regeneration in vivo. Starting with a foundation consisting of an environmentally responsive poly(N-isopropylacrylamide-co-acrylic acid) hydrogel, we incorporated matrix metalloproteinase-13 (MMP-13) degradable crosslinkers and peptides containing integrin-binding domains (i.e., Arg-Gly-Asp) to create a biomimetic matrix designed to encourage osteoblast migration and proliferation. We independently tuned matrix stiffness and peptide concentration to generate a response surface model of osteoblast proliferation on different types of matrices. Osteoblast proliferation was significantly influenced by matrix stiffness (i.e., its complex modulus) and peptide concentration. When implanted in a rat femoral ablation model, these matrices induced bone regeneration only when protease degradable crosslinks were used to create the network. For the matrices with MMP-13 degradable crosslinkers, the bone formed had a trabecular-like structure and was distributed throughout the marrow space. Based on the correlated effects of matrix stiffness and ligand concentration, the response surface model will facilitate improvements in the regenerative capacity of these artificial extracellular matrices.

Background: Gaps at the interface between implant and bone increase the risk of diminished implant fixation and eventual loosening. The purpose of the present study was to determine if combined use of recombinant human transforming growth factor-beta 2 (rhTGF-β2) and bone morphogenetic protein 2 (rhBMP-2) led to greater implant fixation strength in the presence of interface gaps than the use of either growth factor alone. Methods: Twenty-eight skeletally mature adult male dogs received one porous-coated titanium implant in the proximal part of each humerus, for a total of fifty-six implantation sites. Spacers were used to establish an initial 3-mm gap between the implant and the host bone at all fifty-six sites. Forty-two implants were coated with hydroxyapatite-tricalcium phosphate and were used in three growth-factor-treatment groups in which the implants placed in the left humerus were loaded with 12 μg of rhTGF-β2 (Group 1, seven animals), 25 μg of rhBMP-2 (Group 2, seven animals), or 12 μg of rhTGF-β2 combined with 25 μg of rhBMP-2 (Group 3, seven animals). In these animals, the twenty-one implants that were placed in the right humerus were loaded with buffer only to serve as contralateral controls. In Group 4 (seven animals), the implants were not coated with hydroxyapatite-tricalcium phosphate, the gap in the left humerus was lightly packed with autogenous bone graft, and the gap in the right humerus was left empty to serve as a contralateral control. All animals were killed at twenty-eight days. The primary end points included three mechanical variables: fixation strength, interface stiffness, and energy to failure. Secondary end points included bone ingrowth and bone volume and trabecular architecture in the gap and in a region located 2 mm medial to the implantation site. Results: The hydroxyapatite-tricalcium phosphate coating had no effect on implant fixation, bone ingrowth, or bone formation in the 3-mm gap. Individual growth factor treatments led to 2.3 to 3.2-fold increases in fixation strength and stiffness as compared with the values for the contralateral controls (p < 0.05). The combined growth factor treatment led to 5.7-fold increases in fixation strength and stiffness compared with the values for the contralateral controls (p < 0.01). Autogenous bone graft treatment was associated with 4.5 to 6.4-fold increases in implant fixation strength and stiffness as compared with the values for the contralateral controls (p < 0.01). Compared with the relevant contralateral controls, energy to failure was increased 3.5-fold in association with TGF-β2 alone (p < 0.05), 4.5-fold in association with TGF-β2 combined with BMP-2 (p < 0.01), and 2.5-fold in association with autogenous bone-grafting. As much as 63% of the variance in the mechanical end points was associated with variance in bone volume and architecture in the 3-mm gap and in the region of interest located 2 mm medial to the implantation site (p < 0.01). Conclusions: In this animal model, the combined use of TGF-β2 and BMP-2 led to more secure mechanical fixation of the implant than did the use of either growth factor alone and demonstrated results that were similar to those associated with the use of autogenous bone graft. Clinical Relevance: This growth factor cocktail is more useful for enhancing implant fixation than individual growth factor therapy and may provide an alternative strategy to autogenous bone-grafting.

Mandibular ramus height restoration by distraction osteogenesis (DO) is a key procedure in mandibular hypoplasia reconstruction. The objective of this study was to evaluate short-term skeletal changes in the regenerated bone after vertical mandibular ramus DO using a buried distraction device. Eight subadult beagle dogs underwent bilateral vertical mandibular ramus DO. After a 7-day latency period, distraction was performed at a rate of 0.5 mm twice a day for 12 days. Four dogs were killed at 1 month and four dogs at 2 months after the end of distraction. One intact beagle was included as an unoperated control. After sacrifice, micro computed tomography (muCT) and mechanical testing of distracted sites were used to measure bone volume (BV), total volume (TV), and mechanical peak load strength, respectively. The muCT images showed wide variation in the response, with some animals demonstrating considerable bone formation and reconstitution of the canal for the inferior alveolar nerve. Quantitatively, BV was no more than 67% and BV/TV was less than 25% of the intact control, and strength was approximately 33% of the intact control value. The 1 and 2 month values were similar. These results suggest that internal distractors can successfully reconstitute bone but that the regenerated tissue did not regain structural and mechanical characteristics of native bone within the 2 month study period.

In an effort to explore Diffraction Enhanced Imaging of bone tissue, experiments were performed to determine if it was possible to use Diffraction Enhanced Imaging to detect microdamage in bovine cortical bone. Measurements were made at the National Synchrotron Light Source where pre- and post-fatigue rocking curve widths of the bone were studied. The rocking curve widths were then compared. Since no consistent pattern of narrowing or broadening of the rocking curve emerged, it is likely that the ultra-small-angle X-ray scattering present in the bone overshadowed any additional changes to rocking curve caused by microdamage of the bone. Larger bone structures were able to be visualized which suggests that microdamage may be visualized with a higher resolution detector.

The purpose of this study was to determine if acetabular articular cartilage damage occurs in the presence or absence of changes in subchondral plate thickness or porosity and trabecular bone architecture after hip hemiarthroplasty. Eight canines were sacrificed 6 months after receiving unilateral hemiarthroplasties in which a cobalt chrome alloy femoral head was used. The acetabular cartilage, subchondral plate, and trabecular bone were quantitatively evaluated. Although the articular cartilage in the treated hip showed gross and histologic degenerative changes, there were no differences in the treated and contralateral hips in any of the trabecular bone parameters or subchondral plate thickness. However, the subchondral plate porosity was increased 2.6-fold in the treated hip. Therefore, degradation of cartilage can occur in the absence of thickening of the subchondral plate or alterations in the supporting trabecular bone architecture. These observations provide a better understanding of the role that periarticular bone has in the degenerative process after hemiarthoplasty.

Various anabolic agents, including transforming growth factor-beta (TGF-beta), have been shown to enhance intramembranous bone regeneration and strengthen the mechanical connection between implant and host skeleton, a prerequisite for clinical success with orthopedic and dental implants. Mechanisms underlying these observations at the level of the gene have received little attention. A rat model was used to examine levels of gene transcription for 21 "osteogenic" genes by real-time polymerase chain reaction at days 1, 3, 5, 7, 10, 14, and 28 in a control group and a group in which the implant was treated with 1 microg recombinant human TGF-beta2 (n = 42, equally divided among the 2 groups and 7 time points). Genes were chosen to represent three functional categories: (1) growth factors, their receptors and antagonists; (2) bone differentiation markers; and (3) inflammation markers. Examination of the transcription profiles showed that nine genes had up-regulated or down-regulated expression levels without a change in timing and 12 genes had accelerated or delayed expression profiles with or without a concomitant change in maximal or minimal expression. The earliest changes (days 1-3) involved accelerated expression profiles for IGF-1R and VEGF and up-regulation of TGF-beta2, TbetaRI, BMP-2, BMP-7, and Cbfa1. Furthermore, principal components analyses showed that some subsets of genes were co-expressed in both groups, although the temporal relationship of these subsets was altered following growth factor treatment. Thus, in addition to changes in individual transcription profiles, the regulatory connections between sets of co-expressed genes may also be affected by exogenously delivered anabolic agents during bone regeneration.

The aim of the current research was to quantify the changes in gene expression in rat bone marrow derived stromal cells (BMSC) to low intensity pulsed ultrasound (LIPUS) during early time points after the ultrasound application. LIPUS at 1.5 MHz, 30 mW/cm(2) was applied to BMSC for a single 20 min treatment. Real-time PCR was carried out to quantify the expression of early response genes and bone differentiation marker genes 0.5, 1, 3, 6 and 12 h after the end of the LIPUS treatment. Compared with the controls, LIPUS treatment resulted in elevated transient expression of early response genes (c-jun, c-myc, COX-2, Egr-1, TSC-22) as well as the bone differentiation marker genes, osteonectin and osteopontin, at 3 h. This induction of early response genes as well as extracellular matrix genes associated with cell proliferation and differentiation may represent the effect of LIPUS to cells of osteoblastic lineage.

Several previous studies of bone repair have shown 2- to 4-fold increases in bone formation following local delivery of exogenous transforming growth factor-beta (TGF-beta). Here, we use quantitative backscatter electron microscopy to test the effect of TGF-beta1 on mineralization of regenerated bone by examining tissue samples from a previously published canine study in which we found increased bone formation. In the experiment, the proximal humeri of 10 male canines were implanted bilaterally for 28 days with porous-coated implants in the presence of a 3 mm gap between the surface of the implant and the host bone. Implants placed in the left humeri were treated with TGF-beta1 at a dose of either 120 microg (n = 5) or 335 microg (n = 5), and the implants placed in the contralateral humeri served as untreated controls. Quantitative backscatter scanning electron microscopy was used to assess the volume fraction of bone and its degree of mineralization in the 3 mm gaps. The calibrated grayscale mean and median values were depressed compared to the controls in the high dose group (p = 0.048 and p = 0.041, respectively), suggesting that high dose TGF-beta delayed or inhibited mineralization of newly formed osteoid.

Articular cartilage is a thin heterogeneous tissue (typical thickness of less than 2 mm) composed of collagen, proteoglycan, and water that coats skeletal joint surfaces. Its physical and chemical integrity are essential for maintaining normal joint articulation. MR imaging techniques are being developed to visualize articular cartilage for the purpose of assessing changes in its structure that occur with age and the onset of joint disease. Emerging high-field MR systems (greater than 4 T) have a higher signal-to-noise ratio, which can be used to improve the spatial resolution for imaging articular cartilage beyond that available in most clinical systems. In this study, we examined the effect of changing the proteoglycan content of articular cartilage on high-resolution (40 × 40 × 100 μm) T1- and T2-weighted images acquired at 11.74 T (500 MHz for protons). Susceptibility artifacts were observed near the cartilage-bone interface that were verified by separating the cartilage from the bone. MR image heterogeneity of articular cartilage was observed as a function of depth and in the transverse directions of the sample. Enzymatic depletion of cartilage proteoglycan demonstrated that the MR image heterogeneity is due in part to variation of the proteoglycan content in different regions of the cartilage. The apparent diffusion coefficient (ADC) was measured as a function of depth in the cartilage. The ADC varied from the subchondral bone to the articular surface for both normal and digested cartilage. In addition, the ADC increased by 20% after 6 hours of enzymatic digestion of proteoglycan. These results suggest a role for high-field MRI of articular cartilage in the management and staging of osteoarthritis and other joint diseases. © 2004 Wiley Periodicals, Inc. Concepts Magn Reson Part B (Magn Reson Engineering) 23B: 33–43, 2004

Rat bone marrow stromal cells were cultured on either Ra (0.14 μm) or Ra (5.8 μm) Ti6Al4V discs for 24 or 48 h. Cells on the Ra (0.14 μm) surface showed typical fibroblastic morphology, whereas cells on the Ra (5.8 μm) surface were in clusters with a more epithelial appearance. RNA was extracted from the cells at both time points, and gene expression was analyzed by using a rat gene microarray. At 24 and 48 h, a similar number of genes were both up- and down-regulated at least twofold on the Ra (5.8 μm) surface compared to the Ra (0.14 μm) surface. We analyzed the relative level of specific groups of genes related to bone and cartilage development, cell adhesion and extracellular matrix proteins, transcription factors, bone morphogenetic proteins, phospholipases, and protein kinases. Roughness did not appear to be a specific stimulator of osteogenesis because genes of both the bone and cartilage lineage were up-regulated on the Ra (5.8 μm) surface. The most prominent change among transcription factors was up-regulation of Hox 1.4 on the Ra (5.8 μm) surface. Up-regulation of phospholipase A2 and SMAD 4 indicate these genes are also involved in the response of cells to an Ra (5.8 μm) surface. Our data show surface roughness alters the expression of a large number of genes in marrow stromal cells, which are related to multiple pathways of mesenchymal cell differentiation. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 70A: 391–401, 2004

Bisphosphonates are emerging as an important treatment for osteoporosis. But whether the reduced fracture risk associated with bisphosphonate treatment is due to increased bone mass, improved trabecular architecture and/or increased secondary mineralization of the calcified matrix remains unclear. We examined the effects of bisphosphonates on both the trabecular architecture and matrix properties of canine trabecular bone. Thirty-six beagles were divided into a control group and two treatment groups, one receiving risedronate and the other alendronate at 5-6 times the clinical dose for osteoporosis treatment. After one year, the dogs were killed, and samples from the first lumbar vertebrae were examined using a combination of micro-computed tomography, finite element modeling, and mechanical testing. By combining these methods, we examined the treatment effects on the calcified matrix and trabecular architecture independently. Conventional histomorphometry and microdamage data were obtained from the second and third lumbar vertebrae of the same dogs [Bone 28 (2001) 524]. Bisphosphonate treatment resulted in an increased apparent Young's modulus, decreased bone turnover, increased calcified matrix density, and increased microdamage. We could not detect any change in the effective Young's modulus of the calcified matrix in the bisphosphonate treated groups. The observed increase in apparent Young's modulus was due to increased bone mass and altered trabecular architecture rather than changes in the calcified matrix modulus. We hypothesize that the expected increase in the Young's modulus of the calcified matrix due to the increased calcified matrix density was counteracted by the accumulation of microdamage.

This study investigated the association between non-invasive measurements of bone mass and markers of dynamic and static hip joint loads in subjects expected to be at peak bone mass. The bone mineral density (BMD) and bone mineral content (BMC) of three proximal femoral sites (neck, greater trochanter, and total) were measured by dual energy X-ray absorptiometry, and the peak external joint moments at the hip during walking and jogging were calculated from gait analyses of 31 normal human subjects ranging in age from 30 to 49 years (18 females, 13 males). Various multiple regression analyses were performed to determine how much of the variance in BMD and BMC was explained by height, body mass, and the peak hip joint moments. In total, the models explained up to 40% of the variance in BMD and 58% of the variance in BMC. Inclusion of height or body mass did not increase the explanatory power of the models for BMD and explained no more than 8% of the total variance in BMC once the joint moments from walking were allowed to enter the models. These data support the hypothesis that variance in peak bone mass is associated with variance in dynamic hip loads largely independent of the effect of static factors such as height and body mass.

The present study demonstrates increased early mechanical fixation of titanium implants coated with a new biomimetic apatite surface in a rat model. Male Sprague- Dawley rats received unilateral femoral medullary implants for periods of 1- 4 weeks. The strength of fixation of the implant to the host bone increased more rapidly in the group receiving apatite-treated implants compared with the control group as evidenced by the apatite group's 21-fold greater fixation strength at 1 week (p 0.009), 4-fold greater fixation strength at 2 weeks (p 0.041), and 2-fold greater fixation strength at 4 weeks (p 0.093) compared with the control. Fixation strength was correlated with bone-implant contact as determined from micro computed tomography assessment of the specimens (r 2 0.338, p 0.011 in the control group and r 2 0.543, p 0.001 in the apatite group). Furthermore, for a given amount of bone-implant contact, the fixation strength was higher in the apatite group than in the control group (p 0.011), suggesting that the bone formed a stronger bond to the apatite coating than to the titanium. This difference in bonding strength accounted for the difference in mechanical behavior. © 2004 Wiley Period- icals, Inc. J Biomed Mater Res 70A: 66 -73, 2004

The unilateral canine model is the most commonly used model of experimental osteoarthritis (OA). In this model, the anterior cruciate ligament (ACL) of one knee is transected and the contralateral joint is usually used as a control. However, dogs, similar to humans, can develop OA spontaneously with old age. Additionally, certain breeds of dogs are genetically predisposed to OA and can develop symptoms at a young age. The goal of this study was to compare the pathological changes of proteoglycans in OA cartilage from dogs that developed OA spontaneously to those that underwent ACL transection. For this reason, biglycan, decorin and fibromodulin levels and degradation patterns were compared by Western blot hybridization, and aggrecan contents were quantified by dimethylmethylene blue assay. The changes in proteoglycan levels in the cartilage of dogs with spontaneous OA, regardless of their age, were very similar to those published for human OA cartilage. However, when OA developed as a result of ACL-surgery, the changes in proteoglycans were different from those of slowly developing spontaneous OA. Therefore, these differences should be taken into consideration when the ACL-transection model is used.

Joint moments are commonly used to characterize gait. Factors like height and weight influence these moments. This study determined which of two commonly used normalization methods, body mass or body weight times height, most reduced the effects of height and weight on peak hip, knee, and ankle external moments during walking. The effectiveness of each normalization method in reducing gender differences was then tested. Gait data from 158 normal subjects were analyzed using unnormalized values, body mass normalized values, and body weight times height normalized values. Without normalization, height or weight accounted for 7-82% of the variance in all 10 peak components of the moments. With normalization, height and weight accounted for at most 6% of the variance with the exception of the hip adduction moment normalized by body weight times height and the ankle dorsiflexion moment normalized by body mass. For the hip adduction moment normalized by body weight times height, height still accounted for 13% of the variance (p