Time course of peri‐implant bone regeneration around loaded and unloaded implants in a rat model

The time‐course of cancellous bone regeneration surrounding mechanically loaded implants affects implant fixation, and is relevant to determining optimal rehabilitation protocols following orthopaedic surgeries. We investigated the influence of controlled mechanical loading of titanium‐coated polyether‐ether ketone (PEEK) implants on osseointegration using time‐lapsed, non‐invasive, in vivo micro‐computed tomography (micro‐CT) scans. Implants were inserted into proximal tibial metaphyses of both limbs of eight female Sprague–Dawley rats. External cyclic loading (60 or 100 μm displacement, 1 Hz, 60 s) was applied every other day for 14 days to one implant in each rat, while implants in contralateral limbs served as the unloaded controls. Hind limbs were imaged with high‐resolution micro‐CT (12.5 μm voxel size) at 2, 5, 9, and 12 days post‐surgery. Trabecular changes over time were detected by 3D image registration allowing for measurements of bone‐formation rate (BFR) and bone‐resorption rate (BRR). At day 9, mean %BV/TV for loaded and unloaded limbs were 35.5 ± 10.0% and 37.2 ± 10.0%, respectively, and demonstrated significant increases in bone volume compared to day 2. BRR increased significantly after day 9. No significant differences between bone volumes, BFR, and BRR were detected due to implant loading. Although not reaching significance (p = 0.16), an average 119% increase in pull‐out strength was measured in the loaded implants. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:997–1006, 2017.

     

Murine Axial Compression Tibial Loading Model to Study Bone Mechanobiology: Implementing the Model and Reporting Results

In vivo, tibial loading in mice is increasingly used to study bone adaptation and mechanotransduction. To achieve standardized and defined experimental conditions, loading parameters and animal-related factors must be considered when performing in vivo loading studies. In this review, we discuss these loading and animal-related experimental conditions, present methods to assess bone adaptation, and suggest reporting guidelines. This review originated from presentations by each of the authors at the workshop “Developing Best Practices for Mouse Models of In Vivo Loading” during the Preclinical Models Section at the Orthopaedic Research Society Annual Meeting, San Diego, CA, March 2017. Following the meeting, the authors engaged in detailed discussions with consideration of relevant literature. The guidelines and recommendations in this review are provided to help researchers perform in vivo loading experiments in mice, and thus further our knowledge of bone adaptation and the mechanisms involved in mechanotransduction. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:233-252, 2020     

Mechanotransduction and functional response of the skeleton to physical stress: The mechanisms and mechanics of bone adaptation

The skeleton's primary mechanical function is to provide rigid levers for muscles to act against as they hold the body upright in defiance of gravity. Many bones are exposed to thousands of repetitive loads each day. During growth and development, the skeleton optimizes its architecture by subtle adaptations to these mechanical loads. The mechanisms for adaptation involve a multistep process of cellular mechanotransduction including: mechanocoupling— conversion of mechanical forces into local mechanical signals, such as fluid shear stresses, that initiate a response by bone cells; biochemical coupling— transduction of a mechanical signal to a biochemical response involving pathways within the cell membrane and cytoskeleton; cell-to-cell signaling from the sensor cells (probably osteocytes and bone lining cells) to effector cells (osteoblasts or osteoclasts) using prostaglandins and nitric oxide as signaling molecules; and effector response— either bone formation or resorption to cause appropriate architectural changes. These architectural changes tend to adjust and improve the bone structure to its prevailing mechanical environment. Structural changes can be predicted, to some extent, by mathematical formulas derived from three fundamental rules: (1) bone adaptation is driven by dynamic, rather than static, loading; (2) extending the loading duration has a diminishing effect on further bone adaptation; (3) bone cells accommodate to a mechanical loading environment, making them less responsive to routine or customary loading signals. Received for publication on Dec. 25, 1997; accepted on Feb. 24, 1998     

Improved anchorage in osteoporotic vertebrae with new implant designs.

The goal of our study was to evaluate two newly developed implant designs and their behavior in terms of subsidence in lumbar vertebral bodies under cyclic loading. The new implants were evaluated in two different configurations (two small prototypes vs. one large prototype with similar load-bearing area) in comparison to a conventional screw-based implant (MACS TL). A pool of 13 spines with a total of 65 vertebrae was used to establish five testing groups of similar bone mineral density (BMD) distribution with eight lumbar vertebrae each. In additional to BMD assessment via dual-energy X-ray absorptiometry, cancellous BMD and structural parameters were determined using a new generation in vivo 3D-pQCT. The specimens were loaded sinusoidally in force control at 1 Hz for 1000 cycles at three load levels (100, 200, and 400 N). A survival analysis using the number of cycles until failure (Cox regression with covariates) was applied to reveal differences between implant groups. All new prototype configurations except the large cylinder survived significantly longer than the control group. The number of cycles until failure was significantly correlated with the structural parameter Tb.Sp. and similarly with the cancellous BMD for three of five implants. In both large prototypes the cycle number until failure significantly correlated with the preoperative distance to the upper endplates. Although the direct relationship between bone structure or density and mechanical breakage behavior cannot be conclusively proven, all the prototypes adapted for poor bone structure performed better than the comparable conventional implant.     

Bone Mass Is Preserved and Cancellous Architecture Altered Due to Cyclic Loading of the Mouse Tibia After Orchidectomy

Introduction : The study of adaptation to mechanical loading under osteopenic conditions is relevant to the development of osteoporotic fracture prevention strategies. We previously showed that loading increased cancellous bone volume fraction and trabecular thickness in normal male mice. In this study, we tested the hypothesis that cyclic mechanical loading of the mouse tibia inhibits orchidectomy (ORX)‐associated cancellous bone loss. Materials and Methods : Ten‐week‐old male C57BL/6 mice had in vivo cyclic axial compressive loads applied to one tibia every day, 5 d/wk, for 6 wk after ORX or sham operation. Adaptation of proximal cancellous and diaphyseal cortical bone was characterized by μCT and dynamic histomorphometry. Comparisons were made between loaded and nonloaded contralateral limbs and between the limbs of ORX (n = 10), sham (n = 11), and basal (n = 12) groups and tested by two‐factor ANOVA with interaction. Results : Cyclic loading inhibited bone loss after ORX, maintaining absolute bone mass at age‐matched sham levels. Relative to sham, ORX resulted in significant loss of cancellous bone volume fraction (?78%) and trabecular number (?35%), increased trabecular separation (67%), no change in trabecular thickness, and smaller loss of diaphyseal cortical properties, consistent with other studies. Proximal cancellous bone volume fraction was greater with loading (ORX: 290%, sham: 68%) than in contralateral nonloaded tibias. Furthermore, trabeculae thickened with loading (ORX: 108%, sham: 48%). Dynamic cancellous bone histomorphometry indicated that loading was associated with greater mineral apposition rates (ORX: 32%, sham: 12%) and smaller percent mineralizing surfaces (ORX: ?47%, sham: ?39%) in the final week. Loading resulted in greater BMC (ORX: 21%, sham: 15%) and maximum moment of inertia (ORX: 39%, sham: 24%) at the cortical midshaft. Conclusions : This study shows that cancellous bone mass loss can be prevented by mechanical loading after hormonal compromise and supports further exploration of nonpharmacologic measures to prevent rapid‐onset osteopenia and associated fractures.     

Functional Adaptation to Loading of a Single Bone Is Neuronally Regulated and Involves Multiple Bones

Regulation of load‐induced bone formation is considered a local phenomenon controlled by osteocytes, although it has also been hypothesized that functional adaptation may be neuronally regulated. The aim of this study was to examine bone formation in multiple bones, in response to loading of a single bone, and to determine whether adaptation may be neuronally regulated. Load‐induced responses in the left and right ulnas and humeri were determined after loading of the right ulna in male Sprague‐Dawley rats (69 ± 16 days of age). After a single period of loading at ?760‐, ?2000‐, or ?3750‐μ? initial peak strain, rats were given calcein to label new bone formation. Bone formation and bone neuropeptide concentrations were determined at 10 days. In one group, temporary neuronal blocking was achieved by perineural anesthesia of the brachial plexus with bupivicaine during loading. We found right ulna loading induces adaptive responses in other bones in both thoracic limbs compared with Sham controls and that neuronal blocking during loading abrogated bone formation in the loaded ulna and other thoracic limb bones. Skeletal adaptation was more evident in distal long bones compared with proximal long bones. We also found that the single period of loading modulated bone neuropeptide concentrations persistently for 10 days. We conclude that functional adaptation to loading of a single bone in young rapidly growing rats is neuronally regulated and involves multiple bones. Persistent changes in bone neuropeptide concentrations after a single loading period suggest that plasticity exists in the innervation of bone.     

Non-invasive axial loading of mouse tibiae increases cortical bone formation and modifies trabecular organization: a new model to study cortical and cancellous compartments in a single loaded element

Systematic study of bones' responses to loading requires simple non-invasive models in appropriate experimental animals where the applied load is controllable and the changes in bone quantifiable. Herein, we validate a model for applying axial loads, non-invasively to murine tibiae. This allows the effects of mechanical loading in both cancellous and cortical bone to be determined within a single bone in which genetic, neuronal and functional influences can also be readily manipulated. Using female C57Bl/J6 mice, peak strains at the tibial mid-shaft were measured during walking (<300 με tension) and jumping (<600 με compression) with single longitudinally oriented strain gauges attached to the bone's lateral and medial surfaces. Identically positioned gauges were also used to determine, for calibration, the strains engendered by external applied compressive tibial loading between the flexed knee and ankle ex vivo. Applied loads between 5 and 13 N produced strains of 1150–2000 με on the lateral surface, and in vivo repetitions of these loads on alternate days for 2 weeks produced significant load magnitude-related increases in cortical bone formation that were similar in mice at 8, 12 and 20 weeks of age. Micro-CT scans showed that loading significantly increases trabecular bone volume in 8 week old mice, but modifies trabecular organization with decreases in trabecular bone volume in 12 and 20 week old mice. This model for loading the tibia has several advantages over other approaches, including scope to study the effects of loading in cancellous as well as cortical bone, against a background of either disuse or of treatment with osteotropic agents within a single bone in normal, mutant and transgenic mice.     

Effect of exercise on tibial and lumbar vertebral bone mass in mature osteopenic rats: Bone histomorphometry study

The effect of moderate running exercise on tibial and lumbar vertebral bone mass was examined in mature osteopenic rats by bone histomorphometry. Ten 37-week-old female Wistar rats, with bone loss resulting from being fed a relatively low-calcium diet for 14 weeks after ovariectomy at the age of 23 weeks, were randomly divided into two groups of five animals each; control and exercise groups. The exercise consisted of treadmill running at 12 m/min for 1 h per day on 5 days per week for 12 weeks. During the exercise period, all animals were fed a standard calcium diet. After 12 weeks of exercise, bone histomorphometry was evaluated for cancellous bone (secondary spongiosa) of the proximal tibia and the fourth lumbar vertebra and for cortical bone of the tibial shaft. The findings suggested that in the mature osteopenic rat, there was a beneficial effect of moderate running exercise with adequate calcium intake on bone mass only in a weight-bearing long bone, the tibia. The mechanism for increased bone mass appeared to be both decreased bone resorption and increased bone formation in cancellous bone and increased bone formation in cortical bone. Received for publication on Dec. 18, 1997; accepted on April 2, 1998     

Efficacy of a sclerostin antibody compared to a low dose of PTH on metaphyseal bone healing

We compared the effect of a sclerostin antibody to that of a clinically relevant dose of parathyroid hormone (PTH) in a rat model for metaphyseal bone healing. Screws of steel or poly methyl methacrylate (PMMA) were inserted bilaterally into the proximal tibia of young male rats. During 4 weeks the animals then received injections of either phosphate buffered saline (control), sclerostin antibody (25 mg/kg, twice weekly) or PTH (5 µg/kg, daily). The healing response around the screws was then assessed by mechanical testing and X‐ray microtomography (µCT). To distinguish between effects on healing and general effects on the skeleton, other untraumatized bone sites and serum biomarkers were also assessed. After 4 weeks of treatment, PTH yielded a 48% increase in screw pull‐out force compared to control (p = 0.03), while the antibody had no significant effect. In contrast, the antibody increased femoral cortical and vertebral strength where PTH had no significant effect. µCT showed only slight changes that were statistically significant for the antibody mainly at cortical sites. The results suggest that a relatively low dose of PTH stimulates metaphyseal repair (screw fixation) specifically, whereas the sclerostin antibody has wide‐spread effects, mainly on cortical bone, with less influence on metaphyseal healing. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:471–476, 2014.     

Perspectives on Bone Mechanical Properties and Adaptive Response to Mechanical Challenge

The bones of the human skeleton serve a mechanical function besides providing a reservoir for calcium and hematopoietic homeostatis. When mechanically challenged, they usually respond and adapt; failure to do so can result in fracture. The mechanical behavior of bone is determined by bone mass and its material properties and by its geometry and architecture. Therefore, in vivo noninvasive measurements of bone mass, geometry, and structure can predict bone strength and are usually employed as a useful-if not always reliable-way to estimate bone fragility, whereas direct bone biomechanical testing in vitro can provide detailed information about mechanical strength. Because bone strains are likely to be regulators of bone mass and strength, exercise protocols designed to counteract the effects of osteoporosis should load the target bone with repeated high peak forces and high strain rates or high impacts on a long-term basis. Such a protocol creates varied strain distributions throughout the bone structure, producing short, repeated strains on the bone in directions to which it is unaccustomed. Exercise in this manner can maintain and perhaps increase bone mass and improve mechanical properties and neuromuscular competency, reducing skeletal fragility and the predisposition to falls.     

Experimental study of vascularized bone grafts in rat: Effect of mechanical loading on bone dynamics

We studied the etiology of postoperative hypertrophy of vascularized bone grafts in a murine experimental model. Syngeneic grafting of revascularized ulna to rat tibia was performed with (group 1) or without (group 2) mechanical loading. The effect of simple overloading on intact bone was studied by segmental resection of the radius (group 3). Bone dynamics were examined by histomorphological measurements. Significant hypertrophy was observed in the early postoperative period in both groups 1 and group 2. After the initial phase, bone growth continued and extensive remodeling was observed in group 1, while marked bone resorption was observed in group 2. Adaptive remodeling was also observed in group 3 after surgery, but was slower than that in groups 1 and 2. Early hypertrophy of vascularized grafts did not correspond to mechanical loading. These results suggest that mechanical loading is the principal factor responsible for remodeling in vascularized bone grafts for their adaptation to a new environment.     

酒精摄入对酒精性骨重构大鼠骨形态、生物力学及骨髓稳态的影响

目的 探讨长期酒精摄入对酒精性骨重构大鼠模型骨组织形态、骨生物力学、骨髓稳态及氧化应激的影响。方法 雄性SD大鼠20只随机分为空白组和酒精组,每组各10只。酒精组大鼠采用20%(vol/vol)酒精腹腔注射,10 mL/kg,每日1次,共12周;正常组给予等体积生理盐水腹腔注射。收集股骨、胫骨和第五椎体行HE和micro-CT扫描评价酒精对骨小梁等骨微观结构的影响;Masson染色评价酒精对胶原纤维蛋白数量及分布的影响;生物力学测试(三点弯曲试验、压缩试验)评价酒精对骨生物力学性能的影响;qRT-PCR法检测骨髓提取及其骨稳态标志物表达情况,评价酒精对COL1A2、BMP2、RANKL和OPG mRNA表达的影响;TBA法/WST-1法检测血清MDA、SOD及SOD/MDA水平。结果 与空白组相比,酒精组骨小梁及胶原纤维蛋白数量减少,骨生物力学性能降低;骨髓COL1A2、BMP2和OPG mRNA表达降低,RANKL mRNA表达增加;血清SOD及SOD/MDA水平降低,而MDA水平增加。结论 酒精可导致骨小梁及胶原纤维蛋白数量减少、生物力学性能降低、骨髓成骨/破骨功能失衡,破坏了机体...     

Early healing of flexor tendon insertion site injuries: Tunnel repair is mechanically and histologically inferior to surface repair in a canine model.

Orthopedic injuries often require surgical reattachment of tendon to bone. Tendon ends can be sutured to bone by direct apposition to the bone surface or by placement within a bone tunnel. Our objective was to compare early healing of a traditional surface versus a novel tunnel method for repair of the flexor digitorum profundus (FDP) tendon insertion site in a canine model. A total of 70 tendon-bone specimens were analyzed 0, 5, 10 or 21 days after injury and repair, using tensile and range of motion mechanical testing, histology and densitometry. Ultimate force (a measure of repair strength) did not differ between surface and tunnel repairs at day 0. Both repair types had reduced strength at 10 and 21 days compared to 0 days, indicative of deterioration of suture grasping strength (tendon softening). At 21 days, tendons repaired in a bone tunnel had 38% lower ultimate force compared to surface repairs (p = 0.017). Histological findings were comparable between repair groups at 5 and 10 days but differed at 21 days, when we saw evidence of maturation of the tendon-bone interface in the surface repairs compared to an immature fibrous interface with no evidence of tendon-bone integration in the tunnel repairs. After accounting for bone removed by the tunnel, no difference in bone mineral density or trabecular bone volume existed between surface and tunnel repairs. If the results of our animal study extend to healing of the human FDP insertion, they indicate that FDP tendons should be reattached to the distal phalanx by suture to the cortical surface rather than suture in a bone tunnel.     

Frequency-dependent enhancement of bone formation in murine tibiae and femora with knee loading

Knee loading is a relatively new loading modality in which dynamic loads are laterally applied to the knee to induce bone formation in the tibia and the femur. The specific aim of the current study was to evaluate the effects of loading frequencies (in Hz) on bone formation at the site away from the loading site on the knee. The left knee of C57/BL/6 mice was loaded with 0.5 N force at 5, 10, or 15 Hz for 3 min/day for 3 consecutive days, and bone histomorphometry was conducted at the site 75% away from the loading site along the length of tibiae and femora. The results revealed frequency-dependent induction of bone formation, in which the dependence was different in the tibia and the femur. Compared with the sham-loading control, for instance, the cross-sectional cortical area was elevated maximally at 5 Hz in the tibia, whereas the most significant increase was observed at 15 Hz in the femur. Furthermore, mineralizing surface, mineral apposition rate, and bone formation rate were the highest at 5 Hz in the tibia (2.0-, 1.4-, and 2.7 fold, respectively) and 15 Hz in the femur (1.5-, 1.2-, and 1.8 fold, respectively). We observed that the tibia had a lower bone mineral density with more porous microstructures than the femur. Those differences may contribute to the observed differential dependence on loading frequencies.     

Osteocyte apoptosis is mechanically regulated and induces angiogenesis in vitro

Osteocyte apoptosis, associated with reduced interstitial fluid flow, precedes osteoclast precursor recruitment and may aid in the delivery of osteoclast precursors to the remodeling site by promoting angiogenesis. To test the association between fluid flow and osteocyte apoptosis, osteocyte‐like MLO‐Y4 cells were subjected to either oscillatory fluid flow (10 dynes/cm², 1 Hz) or no flow conditions with or without TNF‐α treatment to induce osteocyte apoptosis chemically. Flow protected osteocytes from apoptosis regardless of whether they were treated with TNF‐α (p < 0.001) or not (p < 0.05). TNF‐α‐induced apoptotic and nonapoptotic osteocyte conditioned media were used to study the effect of osteocyte apoptosis on angiogenesis. Apoptotic osteocyte conditioned media caused more endothelial cell proliferation (p < 0.05) and migration (p < 0.05), and tubule networks with longer (p < 0.01) and more (p < 0.001) branches than nonapoptotic osteocyte conditioned media. Apoptotic osteocyte conditioned media contained more vascular endothelial growth factor (VEGF) than nonapoptotic osteocyte conditioned media (p < 0.05). VEGF concentrations found in apoptotic osteocyte conditioned media formed endothelial tubule networks with longer (p < 0.05) and more (p < 0.02) branches than VEGF concentrations in nonapoptotic osteocyte conditioned media. Blocking VEGF in apoptotic osteocyte conditioned media abolished tubule formation effects (p < 0.001). Our results suggest that osteocyte apoptosis is flow‐regulated and promotes angiogenesis in a VEGF‐mediated manner. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:523–530, 2011     

Genetic and Environmental Influence on Structural Strength of Weight‐Bearing and Non–Weight‐Bearing Bone: A Twin Study

A bivariate genetic analysis among 217 older female twin pairs showed that, although the structural strength of tibia and radius are mainly regulated by same genetic and environmental factors, the tibia is more affected by environment. Introduction : The habitual loading environment of the bone may modulate the relative contribution of genetic and environmental factors to bone structure. The purpose of this study was to estimate the contribution of the common and site‐specific genetic and environmental factors to interindividual variation in compressive structural strength of the weight‐bearing tibia and non–weight‐bearing radius. Materials and Methods : pQCT scans were obtained from both members of 103 monozygotic (MZ) and 114 dizygotic (DZ) 63‐ to 76‐yr‐old female twin pairs to estimate the compressive strength of the distal tibia and distal radius. Quantitative genetic models were used to decompose the phenotypic variance into additive genetic, shared environmental, and individual environmental effects at each bone site and to study whether these bone sites share genetic or environmental effects. Results : The MZ and DZ twins did not differ in mean age, height, weight, or bone structural strength. The age‐adjusted Cholesky model showed that additive genetic factors accounted for 83% (95% CI, 77–88%) of the variance in radial strength and 61% (95% CI, 52–69%) of the variance in tibial strength, and these were fully correlated. A shared environmental factor accounted for 15% (95% CI, 10–20%) of tibial strength. An individual environmental factor accounted for 17% (95% CI, 12–23%) of the variance in radial strength and 10% (95% CI, 5–17%) of the variance in tibial strength. The relative contribution of an individual environmental factor specific to tibial strength was 14% (95% CI, 11–18%). Conclusions : The results suggest that, in older women, the majority of the individual differences in the compressive structural strength of the forearm and leg are regulated by genetic and environmental factors that are common to both bone sites. However, the relative importance of environmental factors was greater for the weight‐bearing tibia than for the non–weight‐bearing radius. Thus, the heritability of bone strength seems to vary between skeletal sites according to differences in the typical loading environment.