Intramembranous bone regeneration and implant placement using mechanical femoral marrow ablation: rodent models.

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.

Long-term implant fixation and stress-shielding in total hip replacement.

Implant fixation implies a strong and durable mechanical bond between the prosthetic component and host skeleton. Assuming the short-term impediments to implant fixation are successfully addressed and that longer-term issues such as late infection and mechanical failure of the components are avoided, the biological response of the host tissue to the presence of the implant is critical to long-term success. In particular, maintenance of adequate peri-prosthetic bone stock is a key factor. Two major causes of bone loss in the supporting bone are adverse bone remodeling in response to debris shed from the implant and stress-shielding. Here, I review some of the major lessons learned from studying stress-shielding-induced bone loss. It is well known that stress-shielding can be manipulated by altering implant design, but less well appreciated that the development of bone anabolic agents may make it possible to reduce the severity of stress-shielding and the associated bone loss by augmenting the host skeleton through the use of locally or systemically delivered agents. In most cases, mechanical, material and biological factors do not act in isolation, emphasizing that it is often not possible to optimize all boundary conditions.

Particle-induced osteolysis is not accompanied by systemic remodeling but is reflected by systemic bone biomarkers.

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.

Pharmacologic augmentation of implant fixation in osteopenic bone.

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.

Initial application of EPIC-µCT to assess mouse articular cartilage morphology and composition: effects of aging and treadmill running.

OBJECTIVE: The current study was undertaken to adapt Equilibrium Partitioning of an Ionic Contrast agent via microcomputed tomography (EPIC-µCT) to mouse articular cartilage (AC), which presents a particular challenge because it is thin (30 µm) and has a small volume (0.2-0.4 mm(3)), meaning there is only approximately 2-4 µg of chondroitin sulfate (CS) glycosaminoglycan per joint surface cartilage. DESIGN: Using 6 µm isotropic voxels and the negatively charged contrast agent ioxaglate (Hexabrix), we optimized contrast agent concentration and incubation time, assessed two methods of tissue preservation (formalin fixation and freezing), examined the effect of ex vivo chondroitinase ABC digestion on X-ray attenuation, assessed accuracy and precision, compared young and skeletally mature cartilage, and determined patterns of degradation in a murine cartilage damage model induced by treadmill running. RESULTS: The optimal concentration of the contrast agent was 15%, formalin fixation was preferred to freezing, and 2 h of incubation was needed to reach contrast agent equilibrium with formalin-fixed specimens. There was good agreement with histologic measurements of cartilage thickness, although µCT over-estimated thickness by 13% (5 µm) in 6-week-old mice. Enzymatic release of 0.8 µg of chondrotin sulfate (about 40% of the total) increased X-ray attenuation by 17%. There was a 15% increase in X-ray attenuation in 14-week-old mice compared to 6-week-old mice (P < 0.001) and this corresponded to 65% decrease in CS content at 14 weeks. The older mice also had reductions of 33% in cartilage thickness and 44% in cartilage volume (P < 0.001). Treadmill running induced a 16% decrease in cartilage thickness (P = 0.012) and a 12% increase in X-ray attenuation (P = 0.006) in 14-week-old mice. CONCLUSION: This technique enables non-destructive visualization and quantification of murine femoral AC in three dimensions with anatomic specificity and should prove to be a useful new tool in studying degeneration of cartilage in mouse models.

Healing of rat femoral segmental defect with bone morphogenetic protein-2: a dose response study.

OBJECTIVE: Use of recombinant human bone morphogenetic protein-2 (rhBMP-2) is becoming a common clinical approach to enhance bone repair. There is little or no information in the literature on the dose of rhBMP-2 required for effective healing of critical-sized defects such as those associated with trauma. In this study, we used a segmental defect model to assess the dose response of rhBMP-2 using quantitative and qualitative endpoints. METHODS: Femoral defects in rats were replaced with absorbable collagen sponges carrying rhBMP-2 (0, 1, 5, 10 or 20 µg; N=5). At 4-weeks new bone formation was assessed using quantitative (radiography and microcomputed tomography) and qualitative (histology and backscattered-SEM) endpoints statistically compared. RESULTS: rhBMP-2 showed increased bridging in the gap. Quantitative evaluation presented a bi-phasic dose response curve. Histological assessment revealed that with rhBMP-2 the defect showed the presence of spongy bone with the trabeculae layered with active osteoblasts and osteoclasts. The density and compactness of the bone varied with the dose of rhBMP-2. CONCLUSIONS: Our findings revealed that all doses of rhBMP-2 result in new bone formation. However, there is an optimum dose of 12 µg of rhBMP-2 for bone repair in this model, above which and below which less stimulation of bone occurs.

Limitations of using micro-computed tomography to predict bone-implant contact and mechanical 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.

Importance of Fungicide Seed Treatment and Environment on Seedling Diseases of Cotton.

The importance of fungicide seed treatments on cotton was examined using a series of standardized fungicide trials from 1993 to 2004. Fungicide seed treatments increased stands over those from seed not treated with fungicides in 119 of 211 trials. Metalaxyl increased stands compared to nontreated seed in 40 of 119 trials having significant fungicide responses, demonstrating the importance of Pythium spp. on stand establishment. Similarly, PCNB seed treatment increased stands compared to nontreated seed for 44 of 119 trials with a significant response, indicating the importance of Rhizoctonia solani in stand losses. Benefits from the use of newer seed treatment chemistries, azoxystrobin and triazoles, were demonstrated by comparison with a historic standard seed treatment, carboxin + PCNB + metalaxyl. Little to no stand improvement was found when minimal soil temperatures averaged 25°C the first 3 days after planting. Stand losses due to seedling pathogens increased dramatically as minimal soil temperatures decreased to 12°C and rainfall increased. The importance of Pythium increased dramatically as minimal soil temperature decreased and rainfall increased, while the importance of R. solani was not affected greatly by planting environment. These multi-year data support the widespread use of seed treatment fungicides for the control of the seedling disease complex on cotton.

Osteogenic differentiation of rat bone marrow stromal cells by various intensities of low-intensity pulsed ultrasound.

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 biomechanical effects of focused muscle training on medial knee loads in OA of the knee: a pilot, proof of concept study.

BACKGROUND: High dynamic loads of the medial knee are associated with tibiofemoral osteoarthritis (OA) severity and progression. The lower extremity acts as an integrated kinetic unit, thus treatments targeting adjacent segments may promote reductions in the loading of a symptomatic knee. This study examined the biomechanical effects of a lower extremity exercise regimen, emphasizing training of hip abductor musculature, on dynamic knee loads in individuals with knee OA. METHODS: Six subjects with medial compartment knee OA participated in a proof of concept study of a four-week exercise program specifically targeting the hip abductor musculature in combination with traditional quadriceps and hamstring training. Assessments included gait analyses to measure the external knee adduction moment, a surrogate marker of medial knee joint loading as well as WOMAC questionnaires and strength evaluations. RESULTS: All subjects demonstrated a decrease in their external knee adduction moment, with an average decrease of 9% (p<0.05) following the exercise intervention. There was a 78% (p<0.05) decrease in WOMAC knee pain scores. CONCLUSIONS: These results suggest that targeting hip, rather than only knee musculature, may represent an effective biomechanically-based treatment option for medial knee OA.

Comparison of diffraction-enhanced computed tomography and monochromatic synchrotron radiation computed tomography of human trabecular bone.

Diffraction-enhanced imaging (DEI) is an x-ray-based medical imaging modality that, when used in tomography mode (DECT), can generate a three-dimensional map of both the apparent absorption coefficient and the out-of-plane gradient of the index of refraction of the sample. DECT is known to have contrast gains over monochromatic synchrotron radiation CT (SRCT) for soft tissue structures. The goal of this experiment was to compare contrast-to-noise ratio (CNR) and resolution in images of human trabecular bone acquired using SRCT with images acquired using DECT. All images were acquired at the National Synchrotron Light Source (Upton, NY, USA) at beamline X15 A at an x-ray energy of 40 keV and the silicon [3 3 3] reflection. SRCT, apparent absorption DECT and refraction DECT slice images of the trabecular bone were created. The apparent absorption DECT images have significantly higher spatial resolution and CNR than the corresponding SRCT images. Thus, DECT will prove to be a useful tool for imaging applications in which high contrast and high spatial resolution are required for both soft tissue features and bone.

Experimental haemophilic arthropathy in a mouse model of a massive haemarthrosis: gross, radiological and histological changes.

Recurrent haemarthrosis results in chronic synovitis and destructive arthropathy. The long-term effect of a single haemorrhage is not known. To investigate the histopathological changes following a single, but major joint haemorrhage, an animal model of massive haemarthrosis without mechanical trauma was developed and is described in this manuscript. The knee joint capsule of mice deficient in coagulation factor VIII or IX and non-haemophilic wild type mice was punctured to induce a one time, but massive haemorrhage. The single joint puncture resulted in acute haemarthrosis in both types of haemophilic mice but not in wild type mice. Subsequent to injury, the changes in the knee joints were analysed using gross, histological and radiographic assessments and compared with the uninjured knee. In addition, a novel imaging modality, micro-computed tomography, was used to document the structural damage to the joint. Our results indicate that the long-term changes classically observed in patients with advanced haemophilic arthropathy are evident following a single massive haemarthrosis. This model will allow a thorough investigation of the pathobiology of blood-induced joint disease and will be useful to test the efficacy of innovative therapeutic strategies to prevent haemophilic synovitis and arthropathy.

Role of Nonbehavioral Factors in Adjusting Long Bone Diaphyseal Structure in Free-ranging Pan troglodytes.

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 Taï Forest (Côte d'Ivoire) National Parks. We compare cross sections at several locations (35%, 50%, 65% diaphyseal lengths). Community comparisons highlight different diaphyseal shapes of Taï 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.

Diffraction enhanced imaging of controlled defects within bone, including bone-metal gaps.

Gap regions between a bone and an implant, whether existing upon insertion or developing over time, can lead to implant failure. Currently, planar x-ray imaging and CT are the most commonly used methods to evaluate the gap region. An alternative to these available clinical imaging modalities could help to better evaluate bone resorption. Previous experiments with diffraction enhanced imaging (DEI) have shown significant contrast advantages over monochromatic synchrotron radiation (SR) imaging. DEI and planar SR radiography images of bone samples with drill holes and gap regions of known geometry were acquired at the NSLS beamline X15A (Upton, NY, USA). The images acquired with DEI show measurable contrast-to-noise gains when compared to the images acquired using SR radiography.

Bone mineral density in the proximal tibia varies as a function of static alignment and knee adduction angular momentum in individuals with medial knee osteoarthritis.

Based on the premise that bone mass and bone geometry are related to load history and that subchondral bone may play a role in osteoarthritis (OA), we sought to determine if static and dynamic markers of knee joint loads explain variance in the medial-to-lateral ratio of proximal tibial bone mineral density (BMD) in subjects with mild and moderate medial knee OA. We utilized two surrogate markers of dynamic load, the peak knee adduction moment and the knee adduction angular momentum, the latter being the time integral of the frontal plane knee joint moment. BMD for medial and lateral regions of the proximal tibial plateau and one distal region in the tibial shaft was measured in 84 symptomatic subjects with Kellgren and Lawrence radiographic OA grades of 2 or 3. Utilizing gait analysis, the peak knee adduction moment (the external adduction moment of greatest magnitude) and the time integral of the frontal plane knee joint moment (the angular momentum) over the entire stance phase as well as for each of the four subdivisions of stance were calculated. The BMD ratio was not significantly different in grade 2 (1.32 +/- 0.27) and grade 3 knees (1.47 +/- 0.40) (P = 0.215). BMD of the tibial shaft was not correlated with any loading parameter or static alignment. Of all the surrogate gait markers of dynamic load, the knee adduction angular momentum in terminal stance explained the most variance (20%) in the medial-to-lateral BMD ratio (adjusted r(2) = 0.196, P < 0.001). The knee adduction angular momentum for the entire stance phase explained 18% of the variance in the BMD ratio (adjusted r(2) = 0.178, P < 0.001), 10% more variance than explained by the overall peak knee adduction moment (adjusted r(2) = 0.081, P < 0.001). 18% of the variance in the BMD ratio was also explained by the knee alignment angle (adjusted r(2) = 0.183, P < 0.001), and the total explanatory power was increased to 22% when the knee adduction angular momentum in terminal stance was added (change in r(2) = 0.041, P < 0.05, total adjusted r(2) = 0.215, P < 0.001). The BMD ratio and its relationship to dynamic and static markers of loading were independent of height, weight, and the body mass index, demonstrating that both dynamic markers of knee loading as well as knee alignment explained variance in the tibial BMD ratio independent of body size.

Additive enhancement of implant fixation following combined treatment with rhTGF-beta2 and rhBMP-2 in a canine model.

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-beta2) 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 microg of rhTGF-beta2 (Group 1, seven animals), 25 microg of rhBMP-2 (Group 2, seven animals), or 12 microg of rhTGF-beta2 combined with 25 microg 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-beta2 alone (p < 0.05), 4.5-fold in association with TGF-beta2 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-beta2 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.

Saline irrigation does not affect bone formation or fixation strength of hydroxyapatite/tricalcium phosphate-coated implants in a rat model.

Intramembranous bone regeneration is critical to implant fixation. In cementless joint replacement (as opposed to cemented joint replacement), saline irrigation is not typically performed during surgery so that the osteogenic stimulus provided by the marrow is preserved. Several groups are now using the rat marrow ablation model to study intramembranous bone regeneration and implant fixation. In this model, the marrow contents are mechanically disrupted, and debris is often cleared by saline irrigation, a step that appears inconsistent with the clinical situation. Furthermore, in contrast to conventional wisdom, it has been reported that saline irrigation enhanced bone-implant contact and peri-implant bone formation in the rat model (Ishizaka et al. Bone 1996;19:589-594), although mechanical fixation of the implant was not investigated. Accordingly, the present study was performed to determine if saline irrigation leads to enhanced mechanical fixation of implants in the rat model. Forty-eight 400 to 450 g male rats were divided equally into two groups. The treatment group, in contrast to the control group, received saline irrigation in the ablated medullary canal prior to placement of hydroxyapatite/tricalcium phosphate-coated implants. Eight animals in each group were killed at 2, 4, or 8 weeks after implantation, at which time the specimens were analyzed by micro computed tomography to measure bone formation around the implant, followed by a mechanical pull-out test to measure the strength of fixation of the implant. As expected, there was increased fixation strength over time, but there were no significant differences in peri-implant bone volume, bone-implant contact, or implant fixation strength between the two groups. Thus, we found no effect of saline irrigation on bone formation or implant fixation strength in this study in which the implant had an osteoconductive coating.

Effect of low intensity pulsed ultrasound and BMP-2 on rat bone marrow stromal cell gene expression.

To determine how low intensity pulsed ultrasound alters gene expression in rat bone marrow stromal cells and to see if combining this stimulation with BMP-2, cells were pre-cultured for eight days in the presence of 50 microg/ml ascorbic acid and then exposed to either low intensity US or 100 ng/ml BMP-2 or both combined, beginning on the first, third fifth or seventh day of culture so that cells were exposed to the stimuli for one, three, five or seven days. Real time PCR was used to determine the effect of these treatments on gene expression of several genes associated with osteogenesis. The expression of some of the genes (Cbfa-1/Runx2, IGF-receptor, Alk-3, alkaline phosphatase, osteopontin, TGF-beta1, BMP-7) was increased compared to untreated controls. Combination of US and BMP-2 treatment did not lead to synergy of the two stimuli. Cbfa-1 stimulation occurred more quickly with US than with BMP-2. Increases in gene expression were greatest after 3 days exposure to US, with similar results for BMP-2 treatment implying that there may be a time dependence for the stimulus of osteogenic gene expression in stromal cells.

Depressed bone mineralization following high dose TGF-beta1 application in an orthopedic implant model.

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.