Osseointegration of functionally graded Ti6Al4V porous implants: Histology of the pore network.

The additive manufacturing of titanium into porous geometries offers a means to generate low-stiffness endosseous implants with a greater surface area available for osseointegration. In this work, selective laser melting was used to produce gyroid-based scaffolds with a uniform pore size of 300 µm or functionally graded pore size from 600 µm to 300 µm. Initial in vitro assessment with Saos-2 cells showed favourable cell proliferation at pore sizes of 300 and 600 µm. Following implantation into rabbit tibiae, early histological observations at four weeks indicated some residual inflammation alongside neovessel infiltration into the scaffold interior and some early apposition of mineralized bone tissue. At twelve weeks, both scaffolds were filled with a mixture of adipocyte-rich marrow, micro-capillaries, and mineralized bone tissue. X-ray microcomputed tomography showed a higher bone volume fraction (BV/TV) and percentage of bone-implant contact (BIC) in the implants with 300 µm pores than in the functionally graded specimens. In functionally graded specimens, localized BV/TV measurement was observed to be higher in the innermost region containing smaller pores (estimated at 300-400 µm) than in larger pores at the implant exterior. The unit cell topology of the porous implant was also observed to guide the direction of bone ingrowth by conducting along the implant struts. These results suggest that in vivo experimentation is necessary alongside parametric optimization of functionally graded porous implants to predict short-term and long-term bone apposition.

Bone Bioelectricity and Bone-Cell Response to Electrical Stimulation: A Review.

It is hypothesized that bone cells can sense mechanical force in the extracellular network via an electrical signal. This has led to the use of electrical stimulation (ES) to improve fracture repair and mitigate bone loss. Although overlap exists in bone maintenance and fracture healing mechanics, the processes involved in both are very different, resulting in dissimilar behaviors from the cells. Osteocytes are the most abundant cell type in bone tissue, and their basic structure and lineage are fairly well understood, but much debate is present regarding their behavior, with even less known about their behavior in electrical environments. A wide range of research exists on cell behavior under different types of ES, but it is difficult to draw conclusions due to the large variance in stimulation parameters, cell types, and origins (locations and species). By exploring behavior of multiple bone-cell types under different forms of ES, as well as mechanical stimulation through fluid flow, we can determine more about cell reactions to stimuli. In turn, a better understanding of cell response has the potential to improve and broaden therapeutic applications of ES for bone healing and bone loss mitigation, and enhance outcomes for osseointegration into implantable medical devices. These require greater understanding of the bone cellular environment from an electrical perspective as well as cellular responses to ES.

Influence of environmental conditions in bovine bone ablation by ultrafast laser.

Ultrafast lasers are promising tools for surgical applications requiring precise tissue cutting. Shallow ablation depth and slow rate as well as collateral damage are common barriers limiting the use of laser in clinical applications. Localized cooling with water and/or air jet is known to reduce collateral thermal damage. We studied the influence of environmental conditions including air, compressed air flow, still water and water jet on ablation depth, ablation rate and surface morphology on bovine bone samples with an 800 nm femtosecond laser. At 15 J/cm2 , no thermal effect was observed by electron microscopy and Raman spectroscopy. The experimental results indicate that environmental conditions play a significant role in laser ablation. The deepest cavity and highest ablation rate were achieved under the compressed air flow condition, which is attributed to debris removal during the ablation process. The shallowest ablation depth and lowest ablation rates were associated with water flushing. For surface morphology, smooth surface and the absence of microcracks were observed under air flow conditions, while rougher surfaces and minor microcracks were observed under other conditions. These results suggest that ultrafast ablation of bone can be more efficient and with better surface qualities if assisted with blowing air jet.

Lifelong intake of flaxseed or menhaden oil to provide varying n-6 to n-3 PUFA ratios modulate bone microarchitecture during growth, but not after OVX in Sprague-Dawley rats.

SCOPE: Skeletal health is a lifelong process impacted by environmental factors, including nutrient intake. The n-3 source and PUFA ratio affect bone health in growing rats, or following ovariectomy (OVX), but no study has investigated the longitudinal effect of PUFA-supplementation throughout these periods of bone development. METHODS AND RESULTS: One-month-old, Sprague-Dawley rats (n = 98) were randomized to receive one of four diets from 1 through 6 months of age. Diets were modified from AIN-93G to contain a varying amount and source of n-3 (flaxseed versus menhaden oil) to provide an n-6 to n-3 ratio of 10:1 or 5:1. At 3 (prior to SHAM or OVX) and 6 months of age, bone microarchitecture of the tibia was quantified using in vivo micro-computed tomography (SkyScan 1176, Bruker microCT). Providing 5:1 (flaxseed) resulted in lower trabecular thickness and medullary area and greater cortical area fraction during growth compared to diets with a 10:1 PUFA ratio, but many of these differences were not apparent following OVX. CONCLUSION: PUFA-supplementation at levels attainable in human diet modulates some bone structure outcomes during periods of growth, but is not an adequate strategy for the prevention of OVX-induced bone loss in rats.

Preliminary testing of a novel bilateral plating technique for treating periprosthetic fractures of the distal femur.

BACKGROUND: Current stabilization methods for periprosthetic fractures of the distal femur are inadequate in achieving fracture fixation, with complication rates as high as 29%. A major contributor to poor outcomes is that these methods rely only on screw purchase in the bone to maintain fracture reduction. We designed, manufactured and evaluated a novel plating method that utilizes the femoral prosthesis to enhance stability for treatment of distal femoral periprosthetic fractures. METHODS: Medial and lateral plates were designed and manufactured based on geometry of a synthetic femur and femoral prosthesis. The two plates were linked via a compression screw and a small tab on each plate that inserted into pre-existing slots on the prosthesis. Mechanical tests (500N compression or 250N anterior directed cantilever bending), were performed on synthetic femurs with simple transverse fractures (3mm gap) just superior to the distal femoral prosthesis that were stabilized using either the prototype plates or a single lateral plate. Translational movements of the fracture site during loading were measured using 3D motion tracking. FINDINGS: With the single lateral plate, the distal fragment experienced a resultant displacement of 0.40mm under cantilever bending and 0.61mm under compression (13% and 20% respectively of fracture gap width). With the bilateral plates, fracture gap motion was significantly reduced to 0.13mm under bending and compression (4.3% of the fracture gap). INTERPRETATION: Our results indicate that a bilateral plating method is capable of improving stabilization of periprosthetic fractures compared to the traditional lateral plating technique.

Ultrafast laser ablation and machining large-size structures on porcine bone.

When using ultrafast laser ablation in some orthopedic applications where precise cutting/drilling is required with minimal damage to collateral tissue, it is challenging to produce large-sized and deep holes using a tightly focused laser beam. The feasibility of producing deep, millimeter-size structures under different ablation strategies is investigated. X-ray computed microtomography was employed to analyze the morphology of these structures. Our results demonstrated the feasibility of producing holes with sizes required in clinical applications using concentric and helical ablation protocols.

Accumulation of bone strontium measured by in vivo XRF in rats supplemented with strontium citrate and strontium ranelate.

Strontium ranelate is an approved pharmacotherapy for osteoporosis in Europe and Australia, but not in Canada or the United States. Strontium citrate, an alternative strontium salt, however, is available for purchase over-the-counter as a nutritional supplement. The effects of strontium citrate on bone are largely unknown. The study's objectives were 1) to quantify bone strontium accumulation in female Sprague Dawley rats administered strontium citrate (N=7) and compare these levels to rats administered strontium ranelate (N=6) and vehicle (N=6) over 8 weeks, and 2) to verify an in vivo X-ray fluorescence spectroscopy (XRF) system for measurement of bone strontium in the rat. Daily doses of strontium citrate and strontium ranelate were determined with the intention to achieve equivalent amounts of elemental strontium. However, post-hoc analyses of each strontium compound conducted using energy dispersive spectrometry microanalysis revealed a higher elemental strontium concentration in strontium citrate than strontium ranelate. Bone strontium levels were measured at baseline and 8 weeks follow-up using a unique in vivo XRF technique previously used in humans. XRF measurements were validated against ex vivo measurements of bone strontium using inductively coupled plasma mass spectrometry. Weight gain in rats in all three groups was equivalent over the study duration. A two-way ANOVA was conducted to compare bone strontium levels amongst the three groups. Bone strontium levels in rats administered strontium citrate were significantly greater (p<0.05) than rats administered strontium ranelate and vehicle. ANCOVA analyses were performed with Sr dose as a covariate to account for differences in strontium dosing. The ANCOVA revealed differences in bone strontium levels between the strontium groups were not significant, but that bone strontium levels were still very significantly greater than vehicle.

Angiogenesis is required for stress fracture healing in rats.

Although angiogenesis and osteogenesis are critically linked, the importance of angiogenesis for stress fracture healing is unknown. In this study, mechanical loading was used to create a non-displaced stress fracture in the adult rat forelimb. Fumagillin, an anti-angiogenic agent, was used as the water soluble analogue TNP-470 (25mg/kg) as well as incorporated into lipid-encapsulated α(v)ß(3) integrin targeted nanoparticles (0.25mg/kg). In the first experiment, TNP-470 was administered daily for 5 days following mechanical loading, and changes in gene expression, vascularity, and woven bone formation were quantified. Although no changes in vascularity were detected 3 days after loading, treatment-related downregulation of angiogenic (Pecam1) and osteogenic (Bsp, Osx) genes was observed at this early time point. On day 7, microCT imaging of loaded limbs revealed diminished woven bone formation in treated limbs compared to vehicle treated limbs. In the second experiment, α(v)ß(3) integrin targeted fumagillin nanoparticles were administered as before, albeit with a 100-fold lower dose, and changes in vascularity and woven bone formation were determined. There were no treatment-related changes in vessel count or volume 3 days after loading, although fewer angiogenic (CD105 positive) blood vessels were present in treated limbs compared to vehicle treated limbs. This result manifested on day 7 as a reduction in total vascularity, as measured by histology (vessel count) and microCT (vessel volume). Similar to the first experiment, treated limbs had diminished woven bone formation on day 7 compared to vehicle treated limbs. These results indicate that angiogenesis is required for stress fracture healing, and may have implications for inducing rapid repair of stress fractures.

Porcine cortical bone ablation by ultrashort pulsed laser irradiation.

Ultrashort pulsed lasers in bone ablation show promise for many orthopedic applications. To minimize collateral tissue damage and control the ablation process, the ablation threshold fluence must be well characterized. Using an amplified femtosecond laser (170 fs, 800 nm, 1 kHz), the ablation threshold on unaltered porcine cortical bone was measured using the D(2) method at multiple incident pulse numbers ranging from 25 to 1000 pulses per spot. The lowered threshold at greater pulse numbers indicated an incubation effect. Using a power law model, the incubation coefficient of unaltered porcine cortical bone was found to be 0.89 ± 0.03. Through extrapolation, the single-pulse ablation threshold was found to be 3.29 ± 0.14 J/cm(2).

Stress fracture healing: fatigue loading of the rat ulna induces upregulation in expression of osteogenic and angiogenic genes that mimic the intramembranous portion of fracture repair.

Woven bone is formed in response to fatigue-induced stress fractures and is associated with increased local angiogenesis. The molecular mechanisms that regulate this woven bone formation are unknown. Our objective was to measure the temporal and spatial expression of osteo- and angiogenic genes in woven bone formation in response to increasing levels of fatigue-induced damage. We used the rat forelimb compression model to produce four discrete levels of fatigue damage in the right ulna of 115 male Fischer rats. Rats were killed at 0 (1 h), 1, 3 and 7 days after loading. Using qRT-PCR, we quantified gene expression associated with osteogenesis (BMP2, Msx2, Runx2, Osx, BSP, Osc), cell proliferation (Hist4), and angiogenesis (VEGF, PECAM-1) from the central half of the ulna. The spatial distribution of BMP2, BSP and PCNA was assessed by immunohistochemistry or in situ hybridization in transverse histological sections 1, 4, and 7 mm distal to the ulnar mid-diaphysis. One hour after loading, BMP2 was significantly upregulated in neurovascular structures in the medial ulnar periosteum. Expression of angiogenic markers (VEGF, PECAM-1) increased significantly between Day 0 and 1 and, as with BMP2 expression, remained upregulated through Day 7. While Osx and BSP were upregulated on Day 1, the other osteogenic genes (Msx2, Runx2, Osx, BSP and Osc) were induced on Day 3 in association with the initiation of periosteal woven bone formation and continued through Day 7. The magnitude of osteogenic gene expression, particularly matrix genes (BSP, Osc) was significantly proportional the level of fatigue damage. The woven bone response to fatigue injury is remarkably similar to the "intramembranous" portion of fracture repair - rapid formation of periosteal woven bone characterized by early BMP2 expression, cell proliferation, and upregulation of osteogenic genes. We speculate that woven bone repair of fatigue damage may be an abbreviated fracture response without the requirement for endochondral repair. We conclude that bone fatigue repair is a process similar to intramembranous fracture repair characterized by increases in the expression of genes associated with angiogenesis, cell proliferation and osteoblastogenesis, and that the response from the local vasculature precedes the osteogenic response to fatigue loading.

Damaging fatigue loading stimulates increases in periosteal vascularity at sites of bone formation in the rat ulna.

Bone formation in a variety of contexts depends on angiogenesis; however, there are few reports of the vascular response to osteogenic skeletal loading. We used the rat forelimb compression model to characterize vascular changes after fatigue loading. The right forelimbs of 72 adult rats were loaded cyclically in vivo to one of four displacement levels, to produce four discrete levels of ulnar damage. Rats were killed 3-14 days after loading, and their vasculature was perfused with silicone rubber. Transverse histological sections were cut along the ulnar diaphysis. We quantified vessel number, average vessel area, total vessel area, and bone area. On day 3, we observed a dramatic periosteal expansion near the ulnar midshaft, with significant increases in periosteal vascularity; total vessel area was increased 250-450% (P < 0.001). Vascularity remained elevated on days 7 and 14. Vessel number and average vessel area were not correlated (P = 0.09) and contributed independently to total vascular increases. Bone area was not increased on day 3 but on days 7 and 14 was increased significantly in all displacement groups (P < 0.01) due to periosteal woven bone formation. Vascular and bone changes depended on longitudinal location (P < 0.001), with peak increases 2 mm distal to the midshaft. Vascular and bone changes also depended on displacement level (P < 0.005), with greater increases at higher levels of fatigue displacement. We conclude that skeletal fatigue loading induces a rapid increase in periosteal vascularity, followed by an increase in bone area. The angiogenic-osteogenic response is spatially coordinated and scaled to the level of the mechanical stimulus.

Creatine monohydrate increases bone mineral density in young Sprague-Dawley rats.

INTRODUCTION: Creatine kinase, found in osteoblasts, is an enzyme that is upregulated in response to interventions that enhance bone mass accretion. Creatine monohydrate supplementation can increase fat-free mass in young healthy men and women and can reduce markers of bone breakdown in boys with Duchenne muscular dystrophy. PURPOSE: The objective of this study was to determine the influence of supplementation with creatine monohydrate on bone structure and function in growing rats, to establish a therapeutic model. MATERIALS AND METHODS: Creatine monohydrate (2% w.w.) (CR; N = 16) or standard rat chow (CON; N = 16) was fed to Sprague-Dawley rats beginning at 5 wk of age, for 8 wk. Bone mineral density (BMD) and content (BMC) were assessed using dual-energy x-ray absorptiometry at the beginning and end of the protocol. The rats were sacrificed, and one femur was removed for the determination of mechanical properties. RESULTS: The CR-treated rats showed greater lumbar BMD and femoral bending load at failure compared with the CON rats (P < 0.05). CONCLUSIONS: Together, these data suggest that creatine monohydrate potentially has a beneficial influence on bone function and structure; further investigation is warranted into its effect on bone functional properties and its effects in disorders associated with bone loss.

In vivo skeletal imaging of 18F-fluoride with positron emission tomography reveals damage- and time-dependent responses to fatigue loading in the rat ulna.

The skeletal response to damaging fatigue loading is not fully understood. We used (18)F-fluoride PET to describe the time course of the skeletal response following the creation of increasing levels of in vivo, fatigue-induced damage. The right forelimbs of 40 adult rats were loaded in vivo in cyclic compression to four levels of subfracture, fatigue displacement: 30, 45, 65, or 85% of fracture displacement. Rats were injected with a bone-seeking radionuclide ((18)F-fluoride) on days 0 (4 h), 2, 4, 7, 9, 11, 18, 24, and 30, and imaged using a small animal positron emission tomography (PET) scanner. We quantified fluoride uptake in the central 50% of the right (loaded) and left (control) forelimbs. There were significant increases in fluoride uptake in loaded forelimbs compared to control on day 0 for all displacement groups. Normalized uptake (loaded/control) reached peak levels 4 to 9 days after loading. Normalized uptake depended significantly on the level of fatigue displacement. Normalized uptake increased progressively from the 30 to the 45% displacement level (P < 0.001), and from the 45 to the 65% level (P < 0.001) but did not differ between 65 and 85% (P = 0.41). Histologically, we observed a rapid periosteal response with increased vascularity as early as day 1 and abundant woven bone formation between days 3 and 7. Periosteal and woven bone thicknesses were greater in bones subjected to more fatigue displacement. We conclude that a single bout of fatigue loading leads to a transient increase in the uptake of (18)F-fluoride, that the uptake is in proportion to the level of initial damage and is associated with increased vascularity and woven bone formation in the first week after loading.

Systemic bone formation with weekly PTH administration in ovariectomized rats.

PURPOSE: Weekly subcutaneous administration of 0 (vehicle), 10 and 80 microg/kg doses of human parathyroid hormone (1-34) [PTH (1-34)] were compared based on their capacity to induce systemic formation of bone in 9 month-old ovariectomized (OVX) Sprague-Dawley rats. METHODS: Changes elicited at bone tissue after 4 weeks of treatment were assessed using dual x-ray absorptiometry, micro-computed tomography (microCT), and ashing. RESULTS: The 10 microg/kg dose led to a significant increase (p<0.025) in femoral bone mineral density (BMD) over vehicle- and 80 microg/kg-treated groups. Similarly, structural analysis of the femoral neck trabecular bone by microCT revealed increases in bone volume fraction and trabecular thickness over the pre-treatment baseline, and vehicle- and 80 microg/kg-treated groups. CONCLUSIONS: The data suggest that the weekly administration of 10 microg/kg of PTH (1-34) was sufficient to significantly promote the bone mineral density systemically. The weekly administration of 10 microg/kg over a 4-week treatment period is, to our knowledge, one of the lowest reported total dose of PTH (1-34) shown to induce a net anabolic effect on skeletal tissue in OVX rats.