A Novel Nanostructured Surface on Titanium Implants Increases Osseointegration in a Sheep Model.

BACKGROUND: A nanostructured titanium surface that promotes antimicrobial activity and osseointegration would provide the opportunity to create medical implants that can prevent orthopaedic infection and improve bone integration. Although nanostructured surfaces can exhibit antimicrobial activity, it is not known whether these surfaces are safe and conducive to osseointegration. QUESTIONS/PURPOSES: Using a sheep animal model, we sought to determine whether the bony integration of medical-grade, titanium, porous-coated implants with a unique nanostructured surface modification (alkaline heat treatment [AHT]) previously shown to kill bacteria was better than that for a clinically accepted control surface of porous-coated titanium covered with hydroxyapatite (PCHA) after 12 weeks in vivo. The null hypothesis was that there would be no difference between implants with respect to the primary outcomes: interfacial shear strength and percent intersection surface (the percentage of implant surface with bone contact, as defined by a micro-CT protocol), and the secondary outcomes: stiffness, peak load, energy to failure, and micro-CT (bone volume/total volume [BV/TV], trabecular thickness [Tb.Th], and trabecular number [Tb.N]) and histomorphometric (bone-implant contact [BIC]) parameters. METHODS: Implants of each material (alkaline heat-treated and hydroxyapatite-coated titanium) were surgically inserted into femoral and tibial metaphyseal cancellous bone (16 per implant type; interference fit) and in tibial cortices at three diaphyseal locations (24 per implant type; line-to-line fit) in eight skeletally mature sheep. At 12 weeks postoperatively, bones were excised to assess osseointegration of AHT and PCHA implants via biomechanical push-through tests, micro-CT, and histomorphometry. Bone composition and remodeling patterns in adult sheep are similar to that of humans, and this model enables comparison of implants with ex vivo outcomes that are not permissible with humans. Comparisons of primary and secondary outcomes were undertaken with linear mixed-effects models that were developed for the cortical and cancellous groups separately and that included a random effect of animals, covariates to adjust for preoperative bodyweight, and implant location (left/right limb, femoral/tibial cancellous, cortical diaphyseal region, and medial/lateral cortex) as appropriate. Significance was set at an alpha of 0.05. RESULTS: The estimated marginal mean interfacial shear strength for cancellous bone, adjusted for covariates, was 1.6 MPa greater for AHT implants (9.3 MPa) than for PCHA implants (7.7 MPa) (95% CI 0.5 to 2.8; p = 0.006). Similarly, the estimated marginal mean interfacial shear strength for cortical bone, adjusted for covariates, was 6.6 MPa greater for AHT implants (25.5 MPa) than for PCHA implants (18.9 MPa) (95% CI 5.0 to 8.1; p < 0.001). No difference in the implant-bone percent intersection surface was detected for cancellous sites (cancellous AHT 55.1% and PCHA 58.7%; adjusted difference of estimated marginal mean -3.6% [95% CI -8.1% to 0.9%]; p = 0.11). In cortical bone, the estimated marginal mean percent intersection surface at the medial site, adjusted for covariates, was 11.8% higher for AHT implants (58.1%) than for PCHA (46.2% [95% CI 7.1% to 16.6%]; p < 0.001) and was not different at the lateral site (AHT 75.8% and PCHA 74.9%; adjusted difference of estimated marginal mean 0.9% [95% CI -3.8% to 5.7%]; p = 0.70). CONCLUSION: These data suggest there is stronger integration of bone on the AHT surface than on the PCHA surface at 12 weeks postimplantation in this sheep model. CLINICAL RELEVANCE: Given that the AHT implants formed a more robust interface with cortical and cancellous bone than the PCHA implants, a clinical noninferiority study using hip stems with identical geometries can now be performed to compare the same surfaces used in this study. The results of this preclinical study provide an ethical baseline to proceed with such a clinical study given the potential of the alkaline heat-treated surface to reduce periprosthetic joint infection and enhance implant osseointegration.

Effects of Mild and Moderate Monoclonal Antibody Dose on Inflammation, Bone Loss, and Activation of the Central Nervous System in a Female Collagen Antibody-induced Arthritis Mouse Model.

Induction of severe inflammatory arthritis in the collagen antibody-induced arthritis (CAIA) murine model causes extensive joint damage and pain-like behavior compromising analysis. While mild models are less severe, their reduced, variable penetrance makes assessment of treatment efficacy difficult. This study aimed to compare macroscopic and microscopic changes in the paws, along with central nervous system activation between a mild and moderate CAIA model. Balb/c mice (n=18) were allocated to control, mild, and moderate CAIA groups. Paw inflammation, bone volume (BV), and paw volume (PV) were assessed. Histologically, the front paws were assessed for joint inflammation, cartilage damage, and pre/osteoclast-like cells and the lumbar spinal cord and the periaqueductal gray (PAG) region of the brain for glial reactivity. A moderate CAIA dose induced (1) significantly greater local paw inflammation, inflammatory cell infiltration, and PV; (2) significantly more osteoclast-like cells on the bone surface and within the surrounding soft tissue; and (3) significantly greater glial reactivity within the PAG compared with the mild CAIA model. These findings support the use of a moderate CAIA model (higher dose of monoclonal antibodies with low-dose lipopolysaccharide) to induce more consistent histopathological features, without excessive joint destruction.

Human osteocyte expression of Nerve Growth Factor: The effect of Pentosan Polysulphate Sodium (PPS) and implications for pain associated with knee osteoarthritis.

Pentosan polysulphate sodium (PPS) is a promising therapeutic agent for blocking knee pain in individuals with knee osteoarthritis (KOA). The mode of action of PPS in this context is unknown. We hypothesised that the osteocyte, being the principal cell type in the sub-chondral bone, was capable of expressing the pain mediator Nerve Growth Factor (NGF), and that this may be altered in the presence of PPS. We tested the expression of NGF and the response to PPS in the presence or absence of the proinflammatory cytokine tumour necrosis factor-alpha (TNFα), in human osteocytes. For this we differentiated human primary osteoblasts grown from subchondral bone obtained at primary knee arthroplasty for KOA to an osteocyte-like stage over 28d. We also tested NGF expression in fresh osteocytes obtained by sequential digestion from KOA bone and by immunofluorescence in KOA bone sections. We demonstrate for the first time the production and secretion of NGF/proNGF by this cell type derived from patients with KOA, implicating osteocytes in the pain response in this pathological condition and possibly others. PPS inhibited TNFα-induced levels of proNGF secretion and TNFα induced NGF mRNA expression. Together, this provides evidence that PPS may act to suppress the release of NGF in the subchondral bone to ameliorate pain associated with knee osteoarthritis.

Sex-related differences in the skeletal phenotype of aged vitamin D receptor global knockout mice.

The role of the vitamin D receptor (VDR) in maintaining skeletal health appears to be complex and dependent on the physiological context. Global Vdr deletion in a mouse model (Vdr-/-) results in hypocalcemia, secondary hyperparathyroidism and bone features typical of vitamin D-dependent rickets type II. When weanling Vdr-/- mice are fed a diet containing high levels of calcium, phosphorus and lactose, termed the rescue diet, normalisation of serum calcium, phosphate and parathyroid hormone levels results in prevention of rickets at 10 weeks of age. However, 17 week old male Vdr-/- mice, fed the rescue diet, have been reported as osteopenic due to a decrease in bone formation when compared to wild type mice. We now report confirmation of this finding with further data on the effect of the rescue diet on appendicular and axial skeletal structures in male and female Vdr-/- mice at 26 weeks of age compared to Vdr+/- controls. All Vdr-/- mice were normocalcemic with no evidence of any mineralization defect. However, male Vdr-/- mice exhibited significantly reduced mineral in femoral and vertebral bones when compared to control littermate Vdr+/- mice, consistent with the previously reported data. In contrast, 26-week-old female Vdr-/- mice demonstrated significantly increased femoral trabecular bone volume although there was decreased vertebral trabecular bone volume, similar to males, and femoral cortical bone volume was unchanged. Thus, the Vdr-/- mouse model displays sex- and site-specific differences in skeletal structures with long-term feeding of a rescue diet. Although the global Vdr-/- ablation does not permit the determination of skeletal mechanisms producing these differences, these data confirm skeletal changes even when fed the rescue diet.

Circulating RANKL is inversely related to RANKL mRNA levels in bone in osteoarthritic males.

INTRODUCTION: The relationship of circulating levels of receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) with the expression of these molecules in bone has not been established. The objective of this study was to measure, in humans, the serum levels of RANKL and OPG, and the corresponding levels in bone of mRNA encoding these proteins. METHODS: Fasting blood samples were obtained on the day of surgery from patients presenting for hip replacement surgery for primary osteoarthritis (OA). Intraoperatively, samples of intertrochanteric trabecular bone were collected for analysis of OPG and RANKL mRNA, using real time RT-PCR. Samples were obtained from 40 patients (15 men with age range 50 to 79 years, and 25 women with age range 47 to 87 years). Serum total RANKL and free OPG levels were measured using ELISA. RESULTS: Serum OPG levels increased over the age range of this cohort. In the men RANKL mRNA levels were positively related to age, whereas serum RANKL levels were negatively related to age. Again, in the men serum RANKL levels were inversely related (r = -0.70, P = 0.007) to RANKL mRNA levels. Also in the male group, RANKL mRNA levels were associated with a number of indices of bone structure (bone volume fraction relative to bone tissue volume, specific surface of bone relative to bone tissue volume, and trabecular thickness), bone remodelling (eroded surface and osteoid surface), and biochemical markers of bone turnover (serum alkaline phosphatase and osteocalcin, and urinary deoxypyridinoline). CONCLUSION: This is the first report to show a relationship between serum RANKL and the expression of RANKL mRNA in bone.

Structural and remodeling indices in the cancellous bone of the proximal femur across adulthood.

Fragility fractures, including fractures of the femoral neck, result from reductions in the amount, quality and architecture of bone. However, investigations of the underlying structural changes that might predispose to fracture have been largely limited to skeletal sites that do not fracture, such as the iliac crest (IC). The aim of this study was to use histomorphometry to map changes in the architecture and the static remodeling indices of cancellous bone, as a function of age and sex, in bone samples taken from the intertrochanteric (IT) region of the proximal femur at routine autopsy (18-88 years of age). Bone samples for histology were processed from 10-mm cubes of IT cancellous bone. Histomorphometry was performed using an ocular-mounted 10 x 10 graticule at a magnification of x100. An age-dependent decrease in trabecular bone volume was observed in both females and males, as expected (r=-0.75 and r=-0.63, p<0.001, respectively). The underlying mechanisms for bone turnover appeared to be different between males and females. Thus, while the static index of bone resorption (ES/BV) was positively age-dependent in males and females (p<0.001, p<0.03, respectively), the index of bone formation (OS/BV) correlated positively with age in the female group only (p<0.001 vs. NS). Perhaps reflecting an increase in bone formation in older females, the OS/ES ratio was greater in older females than younger females or males. Surprisingly, while resorption indices increased in older males compared with their younger counterparts, bone formation indices increased only in the older female cohort. The IT region in the proximal femur is adjacent to the site commonly involved in fragility fracture. With the limitation that these results describe cross-sectional data, they provide useful insights into changes in the cancellous bone structure and at the bone surface of females and males over the age range of 20-90 years, at a clinically relevant skeletal site.

Differential gene expression of bone anabolic factors and trabecular bone architectural changes in the proximal femoral shaft of primary hip osteoarthritis patients.

Previous studies have shown a generalised increase in bone mass in patients with osteoarthritis (OA). Using molecular histomorphometry, this study examined the in vivo expression of mRNA encoding bone anabolic factors and collagen type I genes (COL1A1, COL1A2) in human OA and non-OA bone. Bone samples were obtained from the intertrochanteric (IT) region of the proximal femur, a skeletal site distal to the active site of disease, from individuals with hip OA at joint replacement surgery and from autopsy controls. Semi-quantitative reverse transcription-polymerase chain reaction analysis revealed elevated mRNA expression levels of alkaline phosphatase (p < 0.002), osteocalcin (OCN) (p < 0.0001), osteopontin (p < 0.05), COL1A1 (p < 0.0001), and COL1A2 (p < 0.002) in OA bone compared to control, suggesting possible increases in osteoblastic biosynthetic activity and/or bone turnover at the IT region in OA. Interestingly, the ratio of COL1A1/COL1A2 mRNA was almost twofold greater in OA bone compared to control (p < 0.001), suggesting the potential presence of collagen type I homotrimer at the distal site. Insulin-like growth factor (IGF)-I, IGF-II, and transforming growth factor-beta1 mRNA levels were similar between OA and control bone. Bone histomorphometric analysis indicated that OA IT bone had increased surface density of bone (p < 0.0003), increased trabecular number (Tb.N) (p < 0.0003), and decreased trabecular separation (Tb.Sp) (p < 0.0001) compared to control bone. When the molecular and histomorphometric data were plotted, positive associations were observed in the controls for OCN/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) versus bone tissue volume (r = 0.82, p < 0.0007) and OCN/GAPDH versus Tb.N (r = 0.56, p < 0.05) and a negative association was observed for OCN/GAPDH versus Tb.Sp (r = -0.64, p < 0.02). These relationships were not evident in trabecular bone from patients with OA, suggesting that bone regulatory processes leading to particular trabecular structures may be altered in this disease. The finding of differential gene expression, as well as architectural changes and differences in molecular histomorphometric associations between OA and controls, at a skeletal site distal to the active site of joint degeneration supports the concept of generalised involvement of bone in the pathogenesis of OA.

Evidence for reduced bone formation surface relative to bone resorption surface in female femoral fragility fracture patients.

Fragility fractures, including neck of femur fractures, result from reductions in the amount, quality and architecture of bone. The aim of this study was to compare the cancellous bone structure, and static indices of bone turnover, in female patients, who had sustained fragility fracture at the femoral neck, with age-matched females without fragility fracture. Bone samples were taken from the intertrochanteric region of the proximal femur of female patients undergoing hip arthroplasty surgery for a subcapital fragility fracture of the femoral neck (#NOF) or from age-matched female control individuals at routine autopsy. Contiguous bone samples were analyzed for undecalcified histomorphometry and for mRNA expression. The histomorphometric data, which were normally distributed, indicated no difference between the mean values for any of the structural parameters in control and fracture samples. In particular, the bone volume (BV/TV) values were not different and did not change significantly with age in these cohorts of individuals aged >65 years. The static indices of bone turnover, eroded surface (ES/BS) and osteoid surface (OS/BS), were positively correlated with age in the >65-year-old control group (p < 0.055 and p < 0.03, respectively). The median values for these indices were not different between the fracture and control groups. However, both the median and the range of OS/BS values were increased for >65-year-old controls compared with a group of younger females aged <65 years, suggesting an increase in bone formation surface in older females in the proximal femur after 65 years of age. When the data were further interrogated, a reduction in the percentage osteoid surface to eroded surface quotient (OS/ES) was found for the fracture group compared with the age-matched control group suggesting a reduced adaptive modeling drift capability in the fracture group. In contiguous bone samples, increased median values for receptor activator of nuclear factor kappa beta (RANK) and interleukin-6 (IL-6) mRNA expression were observed in the fracture group. Study of cultured human osteoblasts showed that recombinant human IL-6 (rhIL-6) inhibited osteoblast differentiation, as measured by an increase in the immature osteoblast marker, STRO-1 and concomitantly decreased expression of the osteoblast maturation marker, alkaline phosphatase. Importantly, cells cultured in the presence of IL-6 showed significantly less mineral deposition in vitro compared with control cultures. These data suggest that perturbations in bone formation surface, relative to resorption surface, are potentially important in producing bone in the proximal femur with increased propensity to fracture.

Increased expression of IL-6 and RANK mRNA in human trabecular bone from fragility fracture of the femoral neck.

Previous studies have implicated pro-inflammatory cytokines in the bone loss of estrogen deficiency. The aim of this study was to investigate the expression of key regulatory molecules of bone remodeling in the trabecular bone microenvironment in osteoporosis. Bone samples were taken from the intertrochanteric region of the proximal femur of patients undergoing total hip arthroplasty for a subcapital fragility fracture of the femoral neck (#NOF). For comparison, samples were taken from age-matched control individuals at routine autopsy. Expression of RANKL, RANK, osteoprotegerin (OPG), IL-6, IL-11, osteocalcin (OCN), and calcitonin receptor (CTR) messenger RNA (mRNA) species were analyzed and the data were nonparametrically distributed. The median expression of the proresorptive genes, RANK and IL-6, were significantly elevated in the fracture group compared to an age-matched control group (2.2 [1.9-2.9; 25th-75th percentiles] > 1.0 [0.4-2.1], P < 0.03; 3.9 [1.8-6.2] > 0.8 [0.7-1.5], P < 0.002, respectively). In contrast, there were no significant differences in expression of RANKL, OPG, CTR, or OCN mRNA between the #NOF and control groups. The median RANKL/OPG mRNA ratio was significantly greater in hip fracture bone than in bone from controls (4.8 [3.8-7.6] > 3.2 [2.1-4.0], P < 0.05). IL-6 mRNA levels associated strongly with RANKL mRNA levels in the #NOF group (r = 0.77, P < 0.001), but not in the control group. A strong positive association was found between IL-11 mRNA levels and RANKL mRNA levels in the #NOF group (r = 0.81, P < 0.001), consistent with the apparent coordinated regulation of IL-6 and IL-11 in bone samples from the #NOF group (r = 0.93, P < 0.0001). These data suggest a relative increase in the expression of the molecular promoters of osteoclast formation and activity in #NOF bone, which may lead to the imbalance between bone formation and resorption associated with fragility fracture.