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Refereed/Peer-reviewed 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.

Background: Huntington’s disease (HD) is a fatal neurodegenerative autosomal dominant disorder with prevalence of 1 : 20000 that has no effective treatment to date. Translatability of candidate therapeutics could be enhanced by additional testing in large animal models because of similarities in brain anatomy, size, and immunophysiology. These features enable realistic pre-clinical studies of biodistribution, efficacy, and toxicity. Objective and Methods: Here we non-invasively characterized alterations in brain white matter microstructure, neurochemistry, neurological status, and mutant Huntingtin protein (mHTT) levels in cerebrospinal fluid (CSF) of aged OVT73 HD sheep. Results: Similar to HD patients, CSF mHTT differentiates HD from normal sheep. Our results are indicative of a decline in neurological status, and alterations in brain white matter diffusion and spectroscopy metric that are more severe in aged female HD sheep. Longitudinal analysis of aged female HD sheep suggests that the decline is detectable over the course of a year. In line with reports of HD human studies, white matter alterations in corpus callosum correlates with a decline in gait of HD sheep. Moreover, alterations in the occipital cortex white matter correlates with a decline in clinical rating score. In addition, the marker of energy metabolism in striatum of aged HD sheep, shows a correlation with decline of clinical rating score and eye coordination. Conclusion: This data suggests that OVT73 HD sheep can serve as a pre-manifest large animal model of HD providing a platform for pre-clinical testing of HD therapeutics and non-invasive tracking of the efficacy of the therapy.

The study aimed to evaluate the comparative osteoconductivity of three commercially available bone void fillers containing gentamicin with respect to new bone, growth, host tissue response and resorption of the implant material. Defects were created in the cancellous bone of the distal femur and proximal tibia of 12-skeletally mature sheep and filled with three commercially available bone void fillers containing gentamicin (Stimulan-G, Cerament-G, Herafill-G). Peripheral blood was taken pre-operatively and at the time of implantation, as well as at intermittent timepoints following surgery to determine systemic gentamicin levels (5-,15- and 30- minutes, 1, 2, 3, 6, 12, 24, 48- and 72-hours, 3-, 6- and 12-weeks). Decalcified, embedded samples were stained with haematoxylin and eosin (H&E) and used to assess the host tissue response and the formation of new bone in the presence of test implant materials. No adverse reactions were noted at harvest at any time points for any cancellous implantation sites with the various implant materials. Comparative microCT analysis of the Stimulan-G, Cerament-G and Herafill-G test materials revealed a similar increase in bone surface area and volume between animals implanted with Stimulan-G or Cerament-G test materials. Animals implanted with Herafill-G test materials demonstrated the lowest increases in bone volume and surface area of the test materials tested, at levels similar to the negative control sites. By 12-weeks, Stimulan-G defects were completely closed with mature bone and bone marrow whilst the Cerament-G material was still present after 12 weeks by histological examination. In conclusion, this study demonstrated differences in the bone regenerative capacity of a range of bone void fillers in an in vivo setting.

Background Context Degenerative disc disease (DDD) is a common, widespread socioeconomic problem. Appropriate large animal models of DDD are required for improved understanding and to serve as preclinical test beds for therapeutic strategies. Purpose To evaluate the effects of short and medium duration immobilization on the sheep lumbar intervertebral disc (IVD) and facet joints (FJs), and to establish a large animal model for DDD research. Study Design An in vivo sheep model evaluating the effect of short- and medium-term immobilization on disc degeneration. Methods Eighteen sheep were equally randomized into three groups: short-term (6-week) immobilization (n=6), medium-term (26-week) immobilization (n=6), and control (no surgery) (n=6). Immobilization of L3–L4 was achieved with pedicle screw and rod implantation, the IVD was kept intact, and the annulus and end plates were not disrupted. The IVD and FJs were assessed with planar radiography, computerized tomography (CT), magnetic resonance imaging (MRI), pure moment biomechanical testing, and histologic analysis. Results Disc height was reduced for 6- and 26-week immobilization groups. The MRI and histologic analysis demonstrated significant disc degeneration for both immobilized groups compared with control, but no statistical difference was detected between short- and medium-term duration. Progressive degenerative changes in FJs were observed with micro-CT and histologic end points. Immobilization significantly reduced lateral bending and flexion-extension range of motion. Conclusions The mechanical environment set up by immobilization alone is capable of inducing lumbar disc degeneration at both 6 and 26 weeks in sheep. Longer duration immobilization did not advance disc degeneration process beyond of that found with short duration. The present model produces a degenerative disc with intact annulus and without acute injury, more closely representing the scenario common in human disc degeneration. This provides a suitable large animal in vivo model for the evaluation of the new therapies for disc degeneration. Further studies would do well to examine the effect of remobilization after immobilization in this model.

Increasing bone ongrowth and ingrowth of polyether ether ketone (PEEK) interbody fusion devices has the potential to improve clinical outcomes.This study evaluated the in vivo response of promoting new bone growth and bone apposition with NanoMetalene (NM) compared with PEEK alone in a cancellous implantation site with an empty aperture.This is a randomized control animal study.Implants and funding for this study were provided by SeaSpine (60,000 USD). Cylindrical dowels with two apertures were prepared as PEEK with a sub-micron layer of the titanium (NM). The titanium coating was applied over the entire implant (Group 1) or just the apertures (Group 2). Polyether ether ketone implants with no coating served as controls (Group 3). Implants were placed in the cancellous bone of the distal femur or proximal tibia with no graft material placed in the apertures in eight adult sheep. Bone ongrowth to the surface of the implant and ingrowth into the apertures was assessed at 4 and 8 weeks after surgery with micro-computed tomography (CT) and undecalcified histology.The apertures in the implants were notably empty in the PEEK group at 4 and 8 weeks. In contrast, new bone formation into the apertures was found in samples coated with NM even though no graft material was placed into the defect. The bone growing into the aperture tracked along the titanium layer. Apertures with the titanium coating demonstrated significantly more bone by micro-CT qualitative grading compared with PEEK with average bone coverage scores of Group 1 (NM) 1.62±0.89, Group 2 (NM apertures only) 1.62±0.77, and Group 3 (PEEK) 0.43±0.51, respectively, at 4 weeks (p.01) and Group 1 (NM) 1.79±1.19, Group 2 (NM apertures only) 1.98±1.18, and Group 3 (PEEK) 0.69±0.87, respectively, at 8 weeks (p.05). The amount of bone in the apertures (ingrowth) quantified using the volumetric data from the micro-CT supported an overall increase in bone volume inside the apertures with the titanium coating compared with PEEK. Histology showed newly formed woven bone tracked along the surface of the titanium in the apertures. The PEEK interface presented the typical nonreactive fibrous tissue inside the apertures at 4 weeks and some focal contact with bone on the outside at 4 weeks and 8 weeks.Micro-CT and histology demonstrated bone ongrowth to the surfaces coated with NM where the newly formed bone tracked along the thin titanium-coated surfaces. Polyether ether ketone surfaces presented the nonreactive fibrous tissue at the interface as previously reported in preclinical scenarios.

The slinging of sheep is a commonly reported, yet poorly described procedure, which is useful for the advancement of veterinary and medical preclinical research. We have described a novel, flexible and repeatable method of suspending sheep to varying degrees while not affecting mobility and thereby improving animal husbandry and welfare.

Introduction Evidence suggests treatments guided by brain oxygen levels improve patient outcomes following severe traumatic brain injury; however, brain oxygen levels are not routinely monitored as an effective non-invasive method has not been established. We undertook a study, in a sheep model of acute brain injury, to assess a new non-invasive brain oximeter. The monitor uses the principles of pulse oximetry to record a pulse and oxygen levels. Methods We studied 8 sheep. An acute increase in intracranial pressure was induced with an injection of blood into the cranial vault. The temporal changes in the brain oximeter, intracranial pressure and cerebral perfusion pressure were recorded. Simultaneous conventional skin pulse oximetry was also recorded to assess the possible influence of skin blood flow on the brain oximeter signal. Results At baseline, a pulsatile waveform consistent with the brain circulation was obtained in 7 animals. The baseline brain pulse was quite distinct from the simultaneous conventional skin pulse and similar in shape to a central venous pressure waveform. Injection of blood into the cranial vault triggered an immediate increase in intracranial pressure and fall in cerebral perfusion pressure, by 60-s cerebral perfusion pressure recovered. The brain oximeter oxygen levels demonstrated similar changes with an immediate fall and recovery by 60 s. Periods of high intracranial pressure were also associated with high-frequency oscillations in the brain pulse waveform; there was, however, no change in the conventional skin pulse oximeter pulse waveform. Conclusion The brain oximeter detected acute changes in both oxygen levels and the brain pulse waveform following an increase in intracranial pressure levels. The brain oximeter could assist clinicians in the management of acute brain injury., Video abstract Point your SmartPhone at the code above. If you have a QR code reader the video abstract will appear. Or use: https://youtu.be/wbf5tjsC3pw

Global gene delivery to the CNS has therapeutic importance for the treatment of neurological disorders that affect the entire CNS. Due to direct contact with the CNS, cerebrospinal fluid (CSF) is an attractive route for CNS gene delivery. A safe and effective route to achieve global gene distribution in the CNS is needed, and administration of genes through the cisterna magna (CM) via a suboccipital puncture results in broad distribution in the brain and spinal cord. However, translation of this technique to clinical practice is challenging due to the risk of serious and potentially fatal complications in patients. Herein, we report development of a gene therapy delivery method to the CM through adaptation of an intravascular microcatheter, which can be safely navigated intrathecally under fluoroscopic guidance. We examined the safety, reproducibility, and distribution/transduction of this method in sheep using a self-complementary adeno-associated virus 9 (scAAV9)-GFP vector. This technique was used to treat two Tay-Sachs disease patients (30 months old and 7 months old) with AAV gene therapy. No adverse effects were observed during infusion or post-treatment. This delivery technique is a safe and minimally invasive alternative to direct infusion into the CM, achieving broad distribution of AAV gene transfer to the CNS.

As spinal fusions require large volumes of bone graft, different bone graft substitutes are being investigated as alternatives. A subclass of calcium phosphate materials with submicron surface topography has been shown to be a highly effective bone graft substitute. In this work, a commercially available biphasic calcium phosphate (BCP) with submicron surface topography (MagnetOs; Kuros Biosciences BV) was evaluated in an Ovine model of instrumented posterolateral fusion. The material was implanted stand-alone, either as granules (BCPgranules) or as granules embedded within a fast-resorbing polymeric carrier (BCPputty) and compared to autograft bone (AG). Twenty-five adult, female Merino sheep underwent posterolateral fusion at L2-3 and L4-5 levels with instrumentation. After 6, 12, and 26 weeks, outcomes were evaluated by manual palpation, range of motion (ROM) testing, micro-computed tomography, histology and histomorphometry. Fusion assessment by manual palpation 12 weeks after implantation revealed 100% fusion rates in all treatment groups. The three treatment groups showed a significant decrease in lateral bending at the fusion levels at 12 weeks (P < 0.05) and 26 weeks (P < 0.001) compared to the 6 week time-point. Flexion-extension and axial rotation were also reduced over time, but statistical significance was only reached in flexion-extension for AG and BCPputty between the 6 and 26 week time-points (P < 0.05). No significant differences in ROM were observed between the treatment groups at any of the time-points investigated. Histological assessment at 12 weeks showed fusion rates of 75%, 92%, and 83% for AG, BCPgranules and BCPputty, respectively. The fusion rates were further increased 26 weeks postimplantation. Similar trends of bone growth were observed by histomorphometry. The fusion mass consisted of at least 55% bone for all treatment groups 26 weeks after implantation. These results suggest that this BCP with submicron surface topography, in granules or putty form, is a promising alternative to autograft for spinal fusion.

Background context The ideal tissue-engineered solution for any bone graft substitute is to assist in the rapid formation of bone and facilitate fusion. Purpose The present study aims to evaluate this E-BMP-2 (Escherichia coli–derived human bone morphogenetic protein-2) in ovine posterolateral lumbar fusion (PLF) to examine the influence of dose and overall performance in a model with similar graft size and diffusive challenges to the human. Study design/setting In vivo large animal model study. Methods An adult ovine PLF was performed in 30 animals with groups of E-BMP-2 with a beta-tricalcium phosphate (β-TCP) carrier at three different dosages, β-TCP alone, and autograft from the iliac crest. The fusions were assessed by radiography (X-ray and microcomputed tomography), mechanical testing, and hard-tissue histology with bone labels at 6, 8, and 10 weeks along with routine paraffin histology at 12 weeks. Results Results showed increasing new bone and fusion rate with E-BMP-2 dose, whereas β-TCP alone was largely resorbed and did not achieve fusion in this model at 12 weeks. Autograft showed similar grading for the amount of bone between the transverse processes but a lower fusion rate than β-TCP/E-BMP-2 groups. Bone labels revealed new bone formation at all time points for the E-BMP2 groups, whereas the autograft group showed active bone formation at 10 weeks. Beta-tricalcium phosphate displayed reliable incorporation into the decorticated host bone, whereas limited new bone was found between the transverse processes. At the center of the fusion mass, increased E-BMP-2 dose led to increased incorporation of β-TCP by new bone. Conclusions These results suggest that E-BMP-2 was capable of producing posterolateral fusion in the ovine model that is equal to or superior to autologous graft in terms of fusion rate and mechanical strength. E-BMP-2 dose had considerable influence on β-TCP granule resorption.

Background Polyetheretherketone (PEEK) has a wide range of clinical applications but does not directly bond to bone. Bulk incorporation of osteoconductive materials including hydroxyapatite (HA) into the PEEK matrix is a potential solution to address the formation of a fibrous tissue layer between PEEK and bone and has not been tested. Questions/purposes Using in vivo ovine animal models, we asked: (1) Does PEEK-HA improve cortical and cancellous bone ongrowth compared with PEEK? (2) Does PEEK-HA improve bone ongrowth and fusion outcome in a more challenging functional ovine cervical fusion model? Methods The in vivo responses of PEEK-HA Enhanced and PEEK-OPTIMA® Natural were evaluated for bone ongrowth in the form of dowels implanted in the cancellous and cortical bone of adult sheep and examined at 4 and 12 weeks as well as interbody cervical fusion at 6, 12, and 26 weeks. The bone-implant interface was evaluated with radiographic and histologic endpoints for a qualitative assessment of direct bone contact of an intervening fibrous tissue later. Gamma-irradiated cortical allograft cages were evaluated as well. Results Incorporating HA into the PEEK matrix resulted in more direct bone apposition as opposed to the fibrous tissue interface with PEEK alone in the bone ongrowth as well as interbody cervical fusions. No adverse reactions were found at the implant–bone interface for either material. Radiography and histology revealed resorption and fracture of the allograft devices in vivo. Conclusions Incorporating HA into PEEK provides a more favorable environment than PEEK alone for bone ongrowth. Cervical fusion was improved with PEEK-HA compared with PEEK alone as well as allograft bone interbody devices. Clinical Relevance Improving the bone–implant interface with a PEEK device by incorporating HA may improve interbody fusion results and requires further clinical studies.

A segmental tibial defect model in a large animal can provide a basis for testing materials and techniques for use in nonunions and severe trauma. This study reports the rationale behind establishing such a model and its design and conclusions. After ethics approval of the study, aged ewes (older than 5 y; n = 12) were enrolled. A 5-cm mid diaphyseal osteoperiosteal defect was made in the left tibia and was stabilized by using an 8-mm stainless-steel cross-locked intramedullary nail. Sheep were euthanized at 12 wk after surgery and evaluated by using radiography, microCT, and soft-tissue histology techniques. Radiology confirmed a lack of hard tissue callus bridging across the defect. Volumetric analysis based on microCT showed bone growth across the 16.5 cm(3) defect of 1.82 ± 0.94 cm(3). Histologic sections of the bridging tissues revealed callus originating from both the periosteal and endosteal surfaces, with fibrous tissue completing the bridging in all instances. Immunohistochemistry was used to evaluate the quality of the healing response. Clinical, radiographic, and histologic union was not achieved by 12 wk. This model may be effective for the investigation of surgical techniques and healing adjuncts for nonunion cases, where severe traumatic injury has led to significant bone loss.

The healing of critical sized segmental defects is an ongoing clinical problem. No method has achieved pre-eminence. The Masquelet technique is a relatively new innovation involving the induction of a fibrous tissue membrane around the bone defect site taking advantage of the body’s foreign body reaction to the presence of a polymethylmethacrylate (PMMA) spacer. The aim of this study was to investigate the properties and characteristics of this induced membrane and its effectiveness when used in conjunction with allograft or an allograft/autograft mix as filler materials in an ovine critical sized defect model. The resultant induced membrane was found to be effective in containing the graft materials in situ. It was demonstrated to be an organised pseudosynovial membrane which expressed bone morphogenic protein 2 (BMP2), transforming growth factor- beta (TGFβ), vascular endothelial growth factor (VEGF), von Willerbrand factor (vWF), interleukin 6 (IL-6) and interleukin 8 (IL-8). While more new bone growth was evident in the test groups compared to the controls animals at 12 weeks, the volumes were not statistically different and no defects were fully bridged. Of the two graft material groups, the allograft/autograft mix was shown to have a more rapid graft resorption rate than the allograft only group. While the Masquelet technique proved effective in producing a membrane to enclose graft materials, its ability to assist in the healing of critical sized segmental defects when compared to empty controls remained inconclusive.

On activation, platelets secrete an array of growth factors that contribute to bone regeneration. Combining platelet-rich plasma (PRP) with bone graft substitutes has the potential to reduce or replace the reliance on autografts. Lack of standardization and improper use may contribute to the conflicting outcomes reported within both preclinical and clinical investigations using PRP. This study investigates the effect of PRP dose on bone augmentation. Eighty critical-sized defects were created in the cancellous bone of the medial proximal tibia and the distal femur of 20 five-year-old female sheep. The defects were treated with three doses of an autologous thrombin-activated PRP combined with a biphasic calcium phosphate (BCP) or autograft and empty defects. Radiography, micro-computed tomography, histology, histomorphometry, and fluorochrome bone labels were examined at 4 weeks. The empty defects did not spontaneously heal. The highest dose of PRP treatment had a significantly greater micro-CT bone volume/total volume compared with the BCP alone (PRP: 30.6%±1.8%; BCP: 24.5%±0.1%). All doses of PRP treatment were significantly greater than the BCP alone for histomorphometric new bone area (PRP: 14.5%±1.3%; BCP: 9.7%±1.5%) and bone ingrowth depth (PRP: 2288±210 μm; BCP:1151±268 μm). From week 2 onward, PRP had a significant effect on the weekly bone ingrowth compared with BCP; however, autografts had the highest amount of weekly fluorescent bone labeling. PRP induces new bone formation with a dose-dependent response at 4 weeks when used with a BCP in sheep.