A New Method for Creating Impact-Induced Intra-Articular Fractures in a Rabbit Model Induces Severe Post-Traumatic Osteoarthritis.

OBJECTIVES: The objective of this work was to develop a model of intra-articular fracture (IAF) in a rabbit and document the speed and severity of degenerative joint changes after fracture fixation. METHODS: With Institutional Animal Care & Use Committee approval, impact-induced IAFs were created in the distal tibia of 16 New Zealand White rabbits. Fractures were fixed with a plate and screws. Pain and function were monitored at regular postoperative intervals with limb loading analysis. Twelve or 26 weeks after fracture, animals were euthanized for histological assessment of cartilage degeneration and micro-computed tomography analysis of bone histomorphometry. RESULTS: Eleven animals successfully completed the study. Maximum foot force in the fractured limb was 41% ± 21% lower than preoperative values ( P = 0.006) 12 weeks after fracture and remained 25% ± 13% lower ( P = 0.081) after 26 weeks. Cortical bone mineral density in micro-computed tomography images was 34% ± 13% lower 12 weeks after fracture ( P < 0.001) and remained (42% ± 8%) lower 26 weeks after fracture ( P < 0.001). Twelve weeks after fracture, Mankin scores of cartilage degeneration were significantly higher in the medial talus ( P = 0.007), lateral talus ( P < 0.001), medial tibia ( P = 0.017), and lateral tibia ( P = 0.002) of the fractured limb compared with the uninjured contralateral limb. Average Mankin scores in the talus increased from 12 to 26 weeks (5.9 ± 0.9 to 9.4 ± 0.4; P < 0.001 lateral; 5.4 ± 1.8 to 7.8 ± 2.0; P = 0.043 medial), indicating substantial and progressive joint degeneration. CONCLUSIONS: The ankle joint of the New Zealand White rabbit provides the smallest available model of impact-induced IAF that can be treated with clinically relevant techniques and replicates key features of healing and degeneration found in human patients.

Effects of Radiotherapy Upon Bone Structure-Strength Relationships Vary With Sex and Fractionation of Dosing.

Background: Radiotherapy for tumor treatment in or near bones often causes osteopenia and/or osteoporosis, and the resulting increased bone fragility can lead to pathologic fractures. Bone mineral density (BMD) is often used to screen for fracture risk, but no conclusive relationship has been established between BMD and the microstructural/ biomechanical changes in irradiated bone. Understanding the effects of radiation dosing regimen on the bone structure-strength relationship would improve the ability to reduce fracture-related complications resulting from cancer treatment. Methods: Thirty-two C57B6J mice aged 10 - 12 weeks old were randomized to single dose (1 x 25 Gy) and fractionated dose (5 x 5 Gy) irradiation groups. Right hindlimbs were irradiated while the contralateral hindlimbs served as the non-irradiated control. Twelve weeks after irradiation, BMD and bone microstructure were assessed with micro-computed tomography, and mechanical strength/stiffness was assessed with a torsion test. The effects of radiation dosing regimen on bone microstructure and strength were assessed using ANOVA, and bone strength-structure relationships were investigated through correlation analysis of microstructural and mechanical parameters. Results: Fractionated irradiation induced significantly greater losses in BMD in the femur (23% - male mice, p=0.016; 19% - female mice) and the tibia (18% - male mice; 6% - female mice) than the single-dose radiation. The associated reductions in trabecular bone volume (-38%) and trabecular number (-34% to -42%), and the increase in trabecular separation (23% to 29%) were only significant in the male mice with fractionated dosing. There was a significant reduction in fracture torque in the femurs of male (p=0.021) and female (p=0.0017) mice within the fractionated radiation group, but not in the single dose radiation groups. There was moderate correlation between bone microstructure and mechanical strength in the single-dose radiation group (r = 0.54 to 0.73), but no correlation in the fractionated dosing group (r=0.02 to 0.03). Conclusion: Our data indicate more detrimental changes in bone microstructure and mechanical parameters in the fractionated irradiation group compared to the single dose group. This may suggest the potential for protecting bone if a needed therapeutic radiation dose can be delivered in a single session rather than administered in fractions.

Zoledronic Acid Implant Coating Results in Local Medullary Bone Growth.

Osteoarthritis (OA) can necessitate surgical interventions to restore the function of the joint in severe cases. Joint replacement surgery is one of the procedures implemented to replace the damaged joint with prosthetic implants in severe cases of OA. However, after successful implantation, a fraction of OA patients still require revision surgery due to aseptic prosthetic loosening. Insufficient osseointegration is one of the factors that contribute to such loosening of the bone implant, which is commonly made from titanium-based materials. Zoledronic acid (ZA), a potent bisphosphonate agent, has been previously shown to enhance osseointegration of titanium implants. Herein, we fabricated ZA/Ca composites using a reverse microemulsion method and coated them with 1,2-dioleoyl-sn-glycero-3-phosphate monosodium salt (DOPA) to form ZA/Ca/DOPA composites. Titanium alloy screws were subsequently dip-coated with a suspension of the ZA/Ca/DOPA composites and poly(lactic-co-glycolic) acid (PLGA) in chloroform to yield Za/PLGA-coated screws. The coated screws exhibited a biphasic in vitro release profile with an initial burst release within 48 h, followed by a sustained release over 1 month. To assess their performance in vivo, the Za/PLGA screws were then implanted into the tibiae of Sprague-Dawley rats. After 8 weeks, microCT imaging showed new bone growth along the medullary cavity around the implant site, supporting the local release of ZA to enhance bone growth around the implant. Histological staining further confirmed the presence of new mineralized medullary bone growth resembling the cortical bone. Such local medullary growth represents an opportunity for future studies with alternative coating methods to fine-tune the local release of ZA from the coating and enhance complete osseointegration of the implant.

Intra-Articular Adeno-Associated Virus-Mediated Proteoglycan 4 Gene Therapy for Preventing Posttraumatic Osteoarthritis.

Lubricin, a glycoprotein encoded by the proteoglycan 4 (PRG4) gene, is an essential boundary lubricant that reduces friction between articular cartilage surfaces. The loss of lubricin subsequent to joint injury plays a role in the pathogenesis of posttraumatic osteoarthritis. In this study, we describe the development and evaluation of an adeno-associated virus (AAV)-based PRG4 gene therapy intended to restore lubricin in injured joints. The green fluorescent protein (GFP) gene was inserted the PRG4 gene to facilitate tracing the distribution of the transgene product (AAV-PRG4-GFP) in vivo. Transduction efficiency of AAV-PRG4-GFP was evaluated in joint cells, and the conditioned medium containing secreted PRG4-GFP was used for shear loading/friction and viability tests. In vivo transduction of joint tissues following intra-articular injection of AAV-PRG4-GFP was confirmed in the mouse stifle joint in a surgical model of destabilization of the medial meniscus (DMM), and chondroprotective activity was tested in a rabbit anterior cruciate ligament transection (ACLT) model. In vitro studies showed that PRG4-GFP has lubricin-like cartilage-binding and antifriction properties. Significant cytoprotective effects were seen when cartilage was soaked in PRG4-GFP before cyclic shear loading (n = 3). Polymerase chain reaction and confocal microscopy confirmed the presence of PRG4-GFP DNA and protein, respectively, in a mouse DMM (n = 3 per group). In the rabbit ACLT model, AAV-PRG4-GFP gene therapy enhanced lubricin expression (p = 0.001 vs. AAV-GFP: n = 7-14) and protected the cartilage from degeneration (p = 0.014 vs. AAV-GFP: n = 9-10) when treatments were administered immediately postoperation, but efficacy was lost when treatment was delayed for 2 weeks. AAV-PRG4-GFP gene therapy protected cartilage from degeneration in a rabbit ACLT model; however, data from the ACLT model suggest that early intervention is essential for efficacy.

Effects of systemically administered abaloparatide, an osteoanabolic PTHrP analog, as an adjuvant therapy for spinal fusion in rats.

BACKGROUND: Abaloparatide is a parathyroid hormone receptor agonist that increases bone formation and reduces vertebral and nonvertebral fracture risk in women with postmenopausal osteoporosis. Animal studies indicate abaloparatide stimulates vertebral bone formation and enhances bony bridging and biomechanical stability of fracture calluses. AIMS: The current study is evaluating the potential utility for abaloparatide as an adjunct therapy for spinal fusions. MATERIAL AND METHODS: The effects of 14 or 28 days of daily subcutaneous injections of abaloparatide (20 µg/kg/d) or vehicle were evaluated in 32 male Sprague-Dawley rats starting 1 day after noninstrumented posterolateral fusion (PLF) with bone autograft. Fusion mass microarchitecture was analyzed by micro-computed tomography (micro-CT) and serum markers of bone formation and bone resorption were evaluated. Motion segments were scored in a blinded manner as fused or unfused by postmortem radiography and manual palpation. RESULTS: Abaloparatide-treated rats showed higher bone formation (serum osteocalcin) at day 14 and 28 compared with vehicle controls, without increases in the bone resorption marker serum TRACP-5b. Micro-CT showed greater trabecular number in fusion masses from the abaloparatide group vs vehicle controls at day 14. Manual palpation and radiography indicated no fusions in either group at day 14, whereas 25% of vehicle-treated rats and 50% of abaloparatide-treated rats had bilateral fusion at day 28. DISCUSSION AND CONCLUSION: In summary, this rat PLF model showed that abaloparatide treatment was associated with higher levels of the bone formation marker osteocalcin, improved fusion mass architecture, and a non- significant 2-fold higher fusion rate compared with vehicle.

A Multi-Functional Implant Induces Bone Formation in a Diabetic Model.

Patients with diabetes mellitus (DM) have defective healing of bone fractures. It was previously shown that nonviral gene delivery of plasmid DNA (pDNA) that independently encodes bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-2 (FGF-2), acts synergistically to promote bone regeneration in a DM animal model. Additionally, both insulin (INS) and the hormonally active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2 D3 ) (VD3) have independently been shown to play key roles in regulating bone fracture healing in DM patients. However, these individual therapies fail to adequately stimulate bone regeneration, illustrating a need for novel treatment of bone fractures in diabetic patients. Here, the ability of local delivery of INS and VD3 along with BMP-2 and FGF-2 genes is investigated to promote bone formation ectopically in Type-2 diabetic rats. A composite consisting of VD3 and INS is developed that contains poly(lactic-co-glycolic acid) microparticles (MPs) embedded in a fibrin gel surrounded by a collagen matrix that is permeated with polyethylenimine (PEI)-(pBMP-2+pFGF-2) nanoplexes. Using a submuscular osteoinduction model, it is demonstrated that local delivery of INS, VD3, and PEI-(pBMP-2+pFGF-2) significantly improves bone generation compared to other treatments, thusimplicating this approach as a method to promote bone regeneration in DM patients with bone fractures.

In Vivo Testing of a Prototype Intradural Spinal Cord Stimulator in a Porcine Model.

BACKGROUND: Chronic midline low back pain is the number one reason for disability in the United States despite the prolific use of medical and surgical interventions. Notwithstanding the widespread use of epidural spinal cord stimulators (SCSs), there remains a large portion of the population with inadequate pain control thought to be because of the limited volume of stimulated neural tissue. Intradural SCSs represent an underexplored alternative strategy with the potential to improve selectivity, power efficiency, and efficacy. We studied and carried out development of an intradural form of an SCS. Herein we present the findings of in vivo testing of a prototype intradural SCS in a porcine model. METHODS: Six female juvenile pigs underwent surgical investigation. One control animal underwent a laminectomy only, whereas the 5 other animals had implantation of an intradural SCS prototype. One of the prototypes was fully wired to enable acute stimulation and concurrent electromyographic recordings. All animals underwent terminal surgery 3 months postimplantation, with harvesting of the spinal column. Imaging (microcomputed tomography scan) and histopathologic examinations were subsequently performed. RESULTS: All animals survived implantation without evidence of neurologic deficits or infection. Postmortem imaging and histopathologic examination of the spinal column revealed no evidence of spinal cord damage, cerebrospinal fluid fistula formation, abnormal bony overgrowth, or dural defect. Viable dura was present between the intra- and extradural plates of the device. Electromyographic recordings revealed evoked motor units from the stimulator. CONCLUSIONS: Chronically implanted intradural device in the porcine model demonstrated safety and feasibility for translation into humans.

A comprehensive finite element model of surgical treatment for cervical myelopathy.

BACKGROUND: Cervical myelopathy is a common and debilitating chronic spinal cord dysfunction. Treatment includes anterior and/or posterior surgical intervention to decompress the spinal cord and stabilize the spine, but no consensus has been made as to the preferable surgical intervention. The objective of this study was to develop an finite element model of the healthy and myelopathic C2-T1 cervical spine and common anterior and posterior decompression techniques to determine how spinal cord stress and strain is altered in healthy and diseased states. METHODS: A finite element model of the C2-T1 cervical spine, spinal cord, pia, dura, cerebral spinal fluid, and neural ligaments was developed and validated against in vivo human displacement data. To model cervical myelopathy, disc herniation and osteophytes were created at the C4-C6 levels. Three common surgical interventions were then incorporated at these levels. FINDINGS: The finite element model accurately predicted healthy and myelopathic spinal cord displacement compared to motions observed in vivo. Spinal cord strain increased during extension in the cervical myelopathy finite element model. All surgical techniques affected spinal cord stress and strain. Specifically, adjacent levels had increased stress and strain, especially in the anterior cervical discectomy and fusion case. INTERPRETATIONS: This model is the first biomechanically validated, finite element model of the healthy and myelopathic C2-T1 cervical spine and spinal cord which predicts spinal cord displacement, stress, and strain during physiologic motion. Our findings show surgical intervention can cause increased strain in the adjacent levels of the spinal cord which is particularly worse following anterior cervical discectomy and fusion.

Pilot Evaluation of Sheep as In Vivo Model for Cochlear Implantation.

OBJECTIVES: The rise in the use of cochlear implants (CIs) has continued to fuel research aimed at improving surgical approaches and the preservation of residual hearing. Current in vivo models involve small animals not suitable for evaluating full-sized CIs nor are prohibitively expensive nonhuman primates. The objective of this study was to develop and evaluate an in vivo model of cochlear implantation in sheep. METHODS: Eight adult, female sheep were implanted with full-sized CIs from three manufacturers using a retrofacial approach to the round window. Partial electrode insertions were performed to a depth of 10 to 12 mm before closure. Round window electrocochleography (ECoG) and auditory brainstem responses (ABR) were conducted during and after surgery. Following a 30-day implantation, cochleae were explanted and imaged using both x-ray microscopy and histology. RESULTS: The surgery was well tolerated although limited complications were observed in three of eight sheep. Electrode insertions were up to 12 mm before insertion resistance noted. ECoG and ABR responses were reduced postimplantation, reflecting changes in cochlear mechanics due to the presence of the implant, and/or insertion trauma. Histological and radiological image analysis showed the presence of intracochlear fibrosis as well as one instance of tip fold-over. CONCLUSIONS: The use of sheep presents a feasible live-animal model to study cochlear implantations. Full-sized implants as well as surgical techniques can be evaluated on functional outcomes such as ABR and ECoG as well as histological markers for residual hearing including intracochlear fibrosis. Use of this model and surgical approach has potential to evaluate CIs and surgical techniques in both the acute and chronic setting.

Electrospun PLGA and ß-TCP (Rebossis-85) in a Lapine Posterolateral Fusion Model.

Background: Calcium phosphate materials have been employed clinically as bone void fillers for several decades. These materials are most often provided in the form of small, porous granules that can be packed to fill the wide variety of size and shape of bony defects encountered. ReBOSSIS-85 (RB-85) is a synthetic bioresorbable bone void filler for the repair of bone defects with handling characteristics of glass wool-like (or cotton ball-like). The objective of this study is to evaluate the in vivo performance of RB-85 (test material), compared to a commercially available bone void filler, Mastergraft Putty (predicate material), when combined with bone marrow aspirate and iliac crest autograft, in an established posterolateral spine fusion rabbit model. Methods: One hundred fifty skeletally mature rabbits had a single level posterolateral fusion performed. Rabbits were implanted with iliac crest bone graft (ICBG), Mastergraft Putty™ plus ICBG, or one of 4 masses of ReBOSSIS-85 (0.2, 0.3, 0.45, or 0.6 g) plus ICBG. Plain films were taken weekly until euthanasia. Following euthanasia at 4, 8, and 12 weeks, the lumbar spine were tested by manual palpation. Spinal columns in the 12 week group were also subjected to non-destructive flexibility testing. MicroCT and histology were performed on a subset of each implant group at each euthanasia period. Results: Radiographic scoring of the fusion sites indicated a normal healing response in all test groups. Bilateral radiographic fusion rates for all test groups were 0% at 4 weeks; ICBG 43%, Mastergraft Putty 50%, RB-85-0.2g 0%, RB-85-0.3g 13%, RB-85-0.45g 38%, and RB-85-0.6g 63% at 8 weeks; and ICBG 50%, Mastergraft Putty 50%, RB-85-0.2g 0%, RB-85-0.3g 25%, RB-85-0.45g 36%, and RB-85-0.6g 50% at 12 weeks.Spine fusion was assessed by manual palpation of the treated motion segments. At 12 weeks, ICBG, MGP, and RB-85-0.6g were fused mechanically in at least 50% of the rabbits. All groups demonstrated significantly less range of motion in both flexion/extension, lateral bending, and axial rotation compared to normal unfused controls.Histopathology analysis of the fusion masses, in all test groups, indicated an expected normal response of mild inflammation with macrophage and multinucleated giant cell response to the graft material at 4 weeks and resolving by 12 weeks. Regardless of test article, new bone formation and graft resorption increased from 4 to 12 weeks post-op. Conclusions: This animal study has demonstrated the biocompatibility and normal healing features associated with the ReBOSSIS-85 bone graft (test material) when combined with autograft as an extender. ReBOSSIS-85 was more effective when a larger mass of test article was used in this study. Clinical Relevance: ReBOSSIS-85 can be used as an extender negating the need for large amounts of local or iliac crest bone in posterolateral fusions.

The Effect of Pulsed Electromagnetic Field and Combined Magnetic Field Exposure Time on Healing of a Rabbit Tibial Osteotomy.

Background: This study compares effectiveness of two commercially available signals, Pulsed Electromagnetic Field (PEMF) and Combined Magnetic Field (CMF) clinical signals, to stimulate bone healing in rabbit tibial osteotomies. Methods: One millimeter osteotomies in New Zealand White rabbits, stabilized with external fixators, were exposed daily to either signal for 30 minutes, three or six hours. Osteotomized sham controls received no signal exposure. Analyses of torsional strength, periosteal callus area and fracture healing stage demonstrated dose responses to increasing daily exposures to both signals. Results: By 14 days torsional strength increased over shams in the three and six hour-treated groups, significant only for the six hour groups (p<0.05). By 21 days both three and six hour-treated groups were significantly stronger than shams (p<0.05, p<0.005) and the PEMF 30 minute treated group also showed significance (p<0.05). PEMF versus CMF-treated groups were not different at any exposure time. Conclusions: Both CMF and PEMF signals were most effective in this model when used for six hours per day. Clinical Relevance: In this model we demonstrate that though both PEMF and CMF are "bioactive" and promote healing at shorter and longer exposure dosages, there exists an "optimal" threshold effect of 6 hours/day electromagnetic wave stimulation for bone healing.

Biomechanical Analysis of the Cervical Spine Following Disc Degeneration, Disc Fusion, and Disc Replacement: A Finite Element Study.

BACKGROUND: Discectomy and fusion is considered the "gold standard" treatment for clinical manifestations of degenerative disc disease in the cervical spine. However, clinical and biomechanical studies suggest that fusion may lead to adjacent-segment disease. Cervical disc arthroplasty preserves the motion at the operated level and may potentially decrease the occurrence of adjacent segment degeneration. The purpose of this study was to investigate the effect of disc generation, fusion, and disc replacement on the motion, disc stresses, and facet forces on the cervical spine by using the finite element method. METHODS: A validated, intact, 3-dimensional finite element model of the cervical spine (C2-T1) was modified to simulate single-level (C5-C6) and 2-level (C5-C7) degeneration. The single-level degenerative model was modified to simulate both single-level fusion and arthroplasty (total disc replacement [TDR]) using the Bryan and Prestige LP discs. The 2-level degenerative model was modified to simulate a 2-level fusion, 2-level arthroplasty, and single-level disc replacement adjacent to single-level fusion (hybrid). The intact models were loaded by applying a moment of ±2 Nm in flexion-extension, lateral bending, and axial rotation. The motion in each direction was noted and the other modified models were loaded by increasing the moment until the primary C2-T1 motion matched that of the intact (healthy) C2-T1 motion. RESULTS: Both Bryan and Prestige discs preserved motion at the implanted level and maintained normal motions at the adjacent nonoperative levels. A fusion resulted in a decrease in motion at the fused level and an increase in motion at the unfused levels. In the hybrid construct, the TDR (both) preserved motion adjacent to the fusion, thus reducing the demand on the other levels. The disc stresses followed the same trends as motion. Facet forces increased considerably at the index level following a TDR. CONCLUSION: The Bryan and Prestige LP TDRs both preserved motion at the implanted level and maintained normal motion and disc stresses at the adjacent levels. The motion patterns of the spine with a TDR more closely resembled that of the intact spine than those of the degenerative or fused models.

Biomechanical assessment of proximal junctional semi-rigid fixation in long-segment thoracolumbar constructs.

OBJECTIVEProximal junctional kyphosis (PJK) and failure (PJF) are potentially catastrophic complications that result from abrupt changes in stress across rigid instrumented and mobile non-fused segments of the spine (transition zone) after adult spinal deformity surgery. Recently, data have indicated that extension (widening) of the transitional zone via use of proximal junctional (PJ) semi-rigid fixation can mitigate this complication. To assess the biomechanical effectiveness of 3 semi-rigid fixation constructs (compared to pedicle screw fixation alone), the authors performed cadaveric studies that measured the extent of PJ motion and intradiscal pressure changes (ΔIDP).METHODSTo measure flexibility and ΔIDP at the PJ segments, moments in flexion, extension, lateral bending (LB), and torsion were conducted in 13 fresh-frozen human cadaveric specimens. Five testing cycles were conducted, including intact (INT), T10-L2 pedicle screw-rod fixation alone (PSF), supplemental hybrid T9 Mersilene tape insertion (MT), hybrid T9 sublaminar band insertion (SLB1), and hybrid T8/T9 sublaminar band insertion (SLB2).RESULTSCompared to PSF, SLB1 significantly reduced flexibility at the level rostral to the upper-instrumented vertebral level (UIV+1) under moments in 3 directions (flexion, LB, and torsion, p ≤ 0.01). SLB2 significantly reduced motion in all directions at UIV+1 (flexion, extension, LB, torsion, p < 0.05) and at UIV+2 (LB, torsion, p ≤ 0.03). MT only reduced flexibility in extension at UIV+1 (p = 0.02). All 3 constructs revealed significant reductions in ΔIDP at UIV+1 in flexion (MT, SLB1, SLB2, p ≤ 0.02) and torsion (MT, SLB1, SLB2, p ≤ 0.05), while SLB1 and SLB2 significantly reduced ΔIDP in extension (SLB1, SLB2, p ≤ 0.02) and SLB2 reduced ΔIDP in LB (p = 0.05). At UIV+2, SLB2 similarly significantly reduced ΔIDP in extension, LB, and torsion (p ≤ 0.05).CONCLUSIONSCompared to MT, the SLB1 and SLB2 constructs significantly reduced flexibility and ΔIDP in various directions through the application of robust anteroposterior force vectors at UIV+1 and UIV+2. These findings indicate that semi-rigid sublaminar banding can most effectively expand the transition zone and mitigate stresses at the PJ levels of long-segment thoracolumbar constructs.

Ovine model of neuropathic pain for assessing mechanisms of spinal cord stimulation therapy via dorsal horn recordings, von Frey filaments, and gait analysis.

BACKGROUND: It is becoming increasingly important to understand the mechanisms of spinal cord stimulation (SCS) in alleviating neuropathic pain as novel stimulation paradigms arise. PURPOSE: Additionally, the small anatomic scale of current SCS animal models is a barrier to more translational research. METHODS: Using chronic constriction injury (CCI) of the common peroneal nerve (CPN) in sheep (ovine), we have created a chronic model of neuropathic pain that avoids motor deficits present in prior large animal models. This large animal model has allowed us to implant clinical grade SCS hardware, which enables both acute and chronic testing using von Frey filament thresholds and gait analysis. Furthermore, the larger anatomic scale of the sheep allows for simultaneous single-unit recordings from the dorsal horn and SCS with minimal electrical artifact. RESULTS: Detectable tactile hypersensitivity occurred 21 days after nerve injury, with preliminary indications that chronic SCS may reverse it in the painful limb. Gait analysis revealed no hoof drop in the CCI model. Single neurons were identified and discriminated in the dorsal horn, and their activity was modulated via SCS. Unlike previous large animal models that employed a complete transection of the nerve, no motor deficit was observed in the sheep with CCI. CONCLUSION: To our knowledge, this is the first reported large animal model of chronic neuropathic pain which facilitates the study of both acute and chronic SCS using complementary behavioral and electrophysiologic measures. As demonstrated by our successful establishment of these techniques, an ovine model of neuropathic pain is suitable for testing the mechanisms of SCS.

Strontium-releasing fluorapatite glass-ceramic scaffolds: Structural characterization and in vivo performance.

There is increasing interest in biodegradable ceramic scaffolds for bone tissue engineering capable of in situ delivery of ionic species favoring bone formation. Strontium has been shown to be osteogenic, but strontium-containing drugs such as strontium ranelate, used in Europe for the treatment of osteoporosis, are now restricted due to clinical evidence of systemic effects. By doping fluorapatite-based glasses with strontium, we developed ceramic scaffolds with fully interconnected macroporosity and cell size similar to that of cancellous bone, that are also capable of releasing strontium. The crystallization behavior, investigated by XRD and SEM, revealed the formation of akermanite and fluorapatite at the surface of strontium-free glass-ceramic scaffolds, and strontium-substituted fluorapatite at the surface of the strontium-doped scaffolds. At 8 weeks after implantation in a rat calvarial critical size defect, scaffolds doped with the highest amount of strontium led to the highest mineral apposition rate. A significantly higher amount of newly-formed bone was found with the strontium-free glass-ceramic scaffold, and possibly linked to the presence of akermanite at the scaffold surface. We demonstrate by energy dispersive XRF analyses of skull sections that strontium was present in newly formed bone with the strontium-doped scaffolds, while a significant amount of fluorine was incorporated in newly formed bone, regardless of composition or crystallization state. STATEMENT OF SIGNIFICANCE: The present work demonstrates the in vivo action of strontium-containing glass-ceramic scaffolds. These bone graft substitutes are targeted at non load-bearing bone defects. Results show that strontium is successfully incorporated in newly formed bone. This is associated with a significantly higher Mineral Apposition Rate. The benefits of in situ release of strontium are demonstrated. The broader scientific impact of this works builds on the concept of resorbable ceramic scaffolds as reservoirs of ionic species capable of enhancing bone regeneration.

Effects of knockout of the receptor for advanced glycation end-products on bone mineral density and synovitis in mice with intra-articular fractures.

Our group employed the mouse closed intra-articular fracture (IAF) model to test the hypothesis that the innate immune system plays a role in initiating synovitis and post-traumatic osteoarthritis (PTOA) in fractured joints. A transgenic strategy featuring knockout of the receptor for advanced glycation end-products (RAGE -/- ) was pursued. The 42 and 84 mJ impacts used to create fractures were in the range previously reported to cause PTOA at 60 days post-fracture. MicroCT (µCT) was used to assess fracture patterns and epiphyseal and metaphyseal bone loss at 30 and 60 days post-fracture. Cartilage degeneration, synovitis, and matrix metalloproteinase (MMP-3, -13) expression were evaluated by histologic analyses. In wild-type mice, µCT imaging showed that 84 mJ impacts led to significant bone loss at 30 days (p < 0.05), but recovered to normal at 60 days. Bone losses did not occur in RAGE-/- mice. Synovitis was significantly elevated in 84 mJ impact wild-type mice at both endpoints (30 day, p = 0.001; 60 day, p = 0.05), whereas in RAGE-/- mice synovitis was elevated only at 30 days (p = 0.02). Mankin scores were slightly elevated in both mouse strains at 30 days, but not at 60 days. Immunohistochemistry revealed significant fracture-related increases in MMP-3 and -13 expression at 30 days (p < 0.05), with no significant difference between genotypes. These findings indicated that while RAGE -/- accelerated recovery from fracture and diminished synovitis, arthritic changes were temporary and too modest to detect an effect on the pathogenesis of PTOA. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2439-2449, 2018.

Targeting mitochondrial responses to intra-articular fracture to prevent posttraumatic osteoarthritis.

We tested whether inhibiting mechanically responsive articular chondrocyte mitochondria after severe traumatic injury and preventing oxidative damage represent a viable paradigm for posttraumatic osteoarthritis (PTOA) prevention. We used a porcine hock intra-articular fracture (IAF) model well suited to human-like surgical techniques and with excellent anatomic similarities to human ankles. After IAF, amobarbital or N-acetylcysteine (NAC) was injected to inhibit chondrocyte electron transport or downstream oxidative stress, respectively. Effects were confirmed via spectrophotometric enzyme assays or glutathione/glutathione disulfide assays and immunohistochemical measures of oxidative stress. Amobarbital or NAC delivered after IAF provided substantial protection against PTOA at 6 months, including maintenance of proteoglycan content, decreased histological disease scores, and normalized chondrocyte metabolic function. These data support the therapeutic potential of targeting chondrocyte metabolism after injury and suggest a strong role for mitochondria in mediating PTOA.

Influence of 45S5 Bioactive Glass in A Standard Calcium Phosphate Collagen Bone Graft Substitute on the Posterolateral Fusion of Rabbit Spine.

INTRODUCTION: Spinal fusion surgery is an effective but costly treatment for select spinal pathology. Historically iliac crest bone graft (ICBG) has remained the gold standard for achieving successful arthrodesis. Given well-established morbidity autograft harvest, multiple bone graft replacements, void fillers, and extenders have been developed. The objective of this study was to evaluate the in vivo efficacy and safety of two mineralized collagen bone void filler materials similar in composition. Both bone void fillers were composed of hydroxyapatite (HA), tricalcium phosphate (TCP) and bovine collagen. The first test article (Bi-Ostetic bioactive glass foam or "45S5") also contained 45S5 bioactive glass particles while the second test article (Formagraft or "FG") did not. 45S5 and FG were combined with bone marrow aspirate and iliac crest autograft and compared to ICBG in an established posterolateral spine fusion rabbit model. MATERIALS AND METHODS: Sixty-nine mature New Zealand White rabbits were divided into 3 test cohorts: ICBG, 45S5, and FG. A Posterolateral fusion model previous validated was utilized to assess fusion efficacy. The test groups were evaluated for spine fusion rate, new bone formation, graft resorption and inflammatory response using radiographic, µCT, biomechanical and histological endpoints at 4, 8 and 12 weeks following implantation. RESULTS: There were 4 clinical complications unrelated to the graft materials and were evenly split between groups (ICBG graft harvest complications; hind limb mobility, chronic pain) and were euthanized. These omissions did not affect the overall outcome of the study. Radiographic scoring of the fusion sites indicated a normal healing response in all test groups, with no adverse reactions and similar progressions of new bone formation observed over time. All groups demonstrated significantly less range of motion in both flexion/extension and lateral bending compared to normal not-fused controls, which supports fusion results observed in the other endpoints. Fusion occurred earlier in the 45S5 group: ICBG 0%, FG 0%, and 45S5 20% at 4 weeks; ICBG 43%, FG 38%, and 45S5 50% at 8 weeks; and ICBG 50%, FG 56%, and 45S5 56% at 12 weeks. Histopathology analysis of the fusion masses, from each test article and time point, indicated an expected normal response for resorbable calcium phosphate (HA/TCP) and collagen graft material. Mild inflammation with macrophage and multinucleated giant cell response to the graft material was evident in all test groups. DISCUSSION: This study has confirmed the biocompatibility, safety, efficacy and bone healing characteristics of the HA-TCP collagen (with or without 45S5 bioactive glass) composites. The results show that the 3 test groups had equivalent long-term fusion performance and outcome at 12 weeks. However, the presence of 45S5 bioactive glass seemed to accelerate the fusion process as evidenced by the higher fusion rates at 4 and 8 weeks for the HA-TCP-collagen composite containing bioactive glass particles. The results also demonstrate that the HA-TCP-45S5 bioactive glass-collagen composite used as an extender closely mirrors the healing characteristics (i.e. amount and quality of bone) of the 100% autograft group.

Changes in Joint Contact Mechanics in a Large Quadrupedal Animal Model After Partial Meniscectomy and a Focal Cartilage Injury.

Acute mechanical damage and the resulting joint contact abnormalities are central to the initiation and progression of post-traumatic osteoarthritis (PTOA). Study of PTOA is typically performed in vivo with replicate animals using artificially induced injury features. The goal of this work was to measure changes in a joint contact stress in the knee of a large quadruped after creation of a clinically realistic overload injury and a focal cartilage defect. Whole-joint overload was achieved by excising a 5-mm wedge of the anterior medial meniscus. Focal cartilage defects were created using a custom pneumatic impact gun specifically developed and mechanically characterized for this work. To evaluate the effect of these injuries on joint contact mechanics, Tekscan (Tekscan, Inc., South Boston, MA) measurements were obtained pre-operatively, postmeniscectomy, and postimpact (1.2-J) in a nonrandomized group of axially loaded cadaveric sheep knees. Postmeniscectomy, peak contact stress in the medial compartment is increased by 71% (p = 0.03) and contact area is decreased by 35% (p = 0.001); the center of pressure (CoP) shifted toward the cruciate ligaments in both the medial (p = 0.004) and lateral (p = 0.03) compartments. The creation of a cartilage defect did not significantly change any aspect of contact mechanics measured in the meniscectomized knee. This work characterizes the mechanical environment present in a quadrupedal animal knee joint after two methods to reproducibly induce joint injury features that lead to PTOA.

Regeneration of bone using nanoplex delivery of FGF-2 and BMP-2 genes in diaphyseal long bone radial defects in a diabetic rabbit model.

Bone fracture healing impairment related to systemic diseases such as diabetes can be addressed by growth factor augmentation. We previously reported that growth factors such as fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2) work synergistically to encourage osteogenesis in vitro. In this report, we investigated if BMP-2 and FGF-2 together can synergistically promote bone repair in a leporine model of diabetes mellitus, a condition that is known to be detrimental to union. We utilized two kinds of plasmid DNA encoding either BMP-2 or FGF-2 formulated into polyethylenimine (PEI) complexes. The fabricated nanoplexes were assessed for their size, charge, in vitro cytotoxicity, and capacity to transfect human bone marrow stromal cells (BMSCs). Using diaphyseal long bone radial defects in a diabetic rabbit model it was demonstrated that co-delivery of PEI-(pBMP-2+pFGF-2) embedded in collagen scaffolds resulted in a significant improvement in bone regeneration compared to PEI-pBMP-2 embedded in collagen scaffolds alone. This study demonstrated that scaffolds loaded with PEI-(pBMP-2+pFGF-2) could be an effective way of promoting bone regeneration in patients with diabetes.