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.

Saos-2 cells cultured under hypoxia rapidly differentiate to an osteocyte-like stage and support intracellular infection by Staphylococcus aureus.

The intracellular infection of osteocytes represents a clinically important aspect of osteomyelitis. However, few human osteocyte in vitro models exist and the differentiation of immature osteoblasts to an osteocyte stage typically takes at least 4-weeks of culture, making the study of this process challenging and time consuming. The osteosarcoma cell line Saos-2 has proved to be a useful model of human osteoblast to mature osteocyte differentiation. Culture under osteogenic conditions in a standard normoxic (21% O2 ) atmosphere results in reproducible mineralization and acquisition of mature osteocyte markers over the expected 28-35 day culture period. In order to expedite experimental assays, we tested whether reducing available oxygen to mimic concentrations experienced by osteocytes in vivo would increase the rate of differentiation. Cells cultured under 1% O2 exhibited maximal mineral deposition by 14 days. Early (COLA1, MEPE) and mature (PHEX, DMP1, GJA1, SOST) osteocyte markers were upregulated earlier under hypoxia compared to normoxia. Cells differentiated under 1% O2 for 14 days displayed a similar ability to internalize Staphylococcus aureus as day 28 cells grown under normoxic conditions. Thus, low oxygen accelerates Saos-2 osteocyte differentiation, resulting in a useful human osteocyte-like cell model within 14 days.

Vitamin D receptor expression in mature osteoclasts reduces bone loss due to low dietary calcium intake in male mice.

Mature osteoclasts express the vitamin D receptor (VDR) and are able to respond to active vitamin D (1α, 25-dihydroxyvitamin D3; 1,25(OH)2D3) by regulating cell maturation and activity. However, the in vivo consequences of vitamin D signalling directly within functionally mature osteoclasts is only partially understood. To investigate the in vivo role of VDR in mature osteoclasts, conditional deletion of the VDR under control of the cathepsin K promoter (CtskCre/Vdr-/-), was assessed in 6 and 12-week-old mice, either under normal dietary conditions (NormCaP) or when fed a low calcium (0.03 %), low phosphorous (0.08 %) diet (LowCaP). Splenocytes from CtskCre/Vdr-/- mice were co-cultured with MLO-Y4 osteocyte-like cells to assess the effect on osteoclastogenesis. Six-week-old CtskCre/Vdr-/- mice demonstrated a 10 % decrease in vertebral bone volume (p < 0.05), which was associated with increased osteoclast size (p < 0.05) when compared to Vdrfl/fl control mice. Control mice fed a LowCaP diet exhibited extensive trabecular bone loss associated with increased osteoclast surface, number and size (p < 0.0001). Interestingly, CtskCre/Vdr-/- mice fed a LowCaP diet showed exacerbated loss of bone volume fraction (BV/TV%) and trabecular number (Tb.N), by a further 22 % and 21 %, respectively (p < 0.05), suggesting increased osteoclastic bone resorption activity with the loss of VDR in mature osteoclasts under these conditions. Co-culture of CtskCre/Vdr-/- splenocytes with MLO-Y4 cells increased resulting osteoclast numbers 2.5-fold, which were greater in nuclei density and exhibited increased resorption of dentine compared to osteoclasts derived from Vdrfl/fl splenocyte cultures. These data suggest that in addition to RANKL-mediated osteoclastogenesis, intact VDR signalling is required for the direct regulation of the differentiation and activity of osteoclasts in both in vivo and ex vivo settings.

Evidence for altered osteoclastogenesis in splenocyte cultures from VDR knockout mice.

The indirect action of 1α,25(OH)2-vitamin-D3 (1,25D) on the osteoclast through stromal signalling is well established. The role of vitamin D in osteoclasts through direct 1,25D-VDR signalling is less well known. We showed previously that local 1,25D synthesis in osteoclasts modified osteoclastogenesis and osteoclastic resorptive activity. In this study, we hypothesised that osteoclasts lacking VDR expression would display an enhanced resorptive capacity due to the loss of 1,25D signalling. Splenocytes were cultured under osteoclast-differentiating conditions from mice with global deletion of the Vdr gene (VDRKO) and this was compared with age-matched wild-type littermate controls (WT). In VDRKO cultures, osteoclastogenesis was reduced, as indicated by fewer TRAP-positive multinucleated cells at all time points measured (p<0.05) compared to WT levels. However, VDRKO osteoclasts demonstrated greater resorption on a per cell basis than their WT counterparts. VDRKO cultures expressed greatly increased c-Fos mRNA compared to WT. In addition, the ratio of expression of the pro-apoptotic gene Bax to the pro-survival gene Bcl-2 was decreased in VDRKO cultures, implying that these osteoclasts may survive longer than WT osteoclasts. Our data indicate abnormal osteoclastogenesis due to the absence of Vdr expression, consistent with direct effects of vitamin D signalling being important for regulating the maturation and resorptive activities of osteoclasts.

Time dependent loss of trabecular bone in human tibial plateau fractures.

We investigated if time between injury and surgery affects cancellous bone properties in patients suffering tibial plateau fractures (TPF), in terms of structural integrity and gene expression controlling bone loss. A cohort of 29 TPF, operated 1-17 days post-injury, had biopsies from the fracture and an equivalent contralateral limb site, at surgery. Samples were assessed using micro-computed tomography and real-time RT-PCR analysis for the expression of genes known to be involved in bone remodeling and fracture healing. Significant decreases in the injured vs control side were observed for bone volume fraction (BV/TV, -13.5 ± 6.0%, p = 0.011), trabecular number (Tb.N, -10.5 ± 5.9%, p = 0.041) and trabecular thickness (Tb.Th, -4.6 ± 2.5%, p = 0.033). Changes in these parameters were more evident in patients operated 5-17 days post-injury, compared to those operated in the first 4 days post-injury. A significant negative association was found between Tb.Th (r = -0.54, p < 0.01) and BV/TV (r = -0.39, p < 0.05) in relation to time post-injury in the injured limb. Both BV/TV and Tb.Th were negatively associated with expression of key molecular markers of bone resorption, CTSK, ACP5, and the ratio of RANKL:OPG mRNA. These structure/gene expression relationships did not exist in the contralateral tibial plateau of these patients. This study demonstrated that there is a significant early time-dependent bone loss in the proximal tibia after TPF. This bone loss was significantly associated with altered expression of genes typically involved in the process of osteoclastic bone resorption but possibly also bone resorption by osteocytes. The mechanism of early bone loss in such fractures should be a subject of further investigation. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2865-2875, 2018.

Absence of vitamin D receptor in mature osteoclasts results in altered osteoclastic activity and bone loss.

Mature osteoclasts express the vitamin D receptor (VDR) and are able to synthesise and respond to 1,25(OH)2D3 via CYP27B1 enzyme activity. Whether vitamin D signalling within osteoclasts is necessary for the regulation of osteoclastic bone resorption in an in vivo setting is unclear. To determine the requirement for the VDR- and CYP27B1-mediated activity in mature osteoclasts, conditional deletion mouse models were created whereby either Vdr or Cyp27b1 gene was inactivated by breeding either Vdrfl/fl or Cyp27b1fl/fl mice with Cathepsin K-Cre transgenic mice (CstkCre) to generate CtskCre/Vdr-/- and CtskCre/Cyp27b1-/- mice respectively. To account for potential CtskCre-meaited off-target deletion of Vdr, Dmp1Cre were also used determine the effect of Vdr deletion in osteocytes. Furthermore, CtskCre/Vdr-/- mice were ovariectomised (OVX) to assess the role of VDR in osteoclasts under bone-loss conditions and bone marrow precursor cells were cultured under osteoclastogenic conditions to assess osteoclast formation. Six-week-old CtskCre/Vdr-/- female mice demonstrated a 15% decrease in femoral BV/TV (p<0.05). In contrast, BV/TV remained unchanged in CtskCre/Cyp27b1-/- mice as well as in Dmp1Cre/VDR-/- mice. When CtskCre/Vdr-/- mice were subjected to OVX, the bone loss that occurred in CtskCre/Vdr-/- was predominantly due to a diminished volume of thinner trabeculae when compared to control levels. These changes in bone volume in CtskCre/Vdr-/- mice occurred without an observable histological change in osteoclast numbers or size. However, while cultured bone marrow-derived osteoclasts from CtskCre/Vdr-/- mice were marginally increased when compared to VDRfl/fl mice, elevated expression of genes such as Cathepsin K, Nfatc1 and VATPase was observed. Collectively, these data indicate that the absence of VDR in mature osteoclasts causes exacerbated bone loss in young mice and during OVX which is associated with enhanced osteoclastic activity and without increased osteoclastogenesis.

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.