Emily B. Petersen

Effects of direct current electrical stimulation on gene expression of osteopromotive factors in a posterolateral spinal fusion model.

Study Design. An in vivo model was used to determine levels of mRNA expression in response to direct current (DC) electrical stimulation in a rabbit posterolateral fusion model. Objectives. This study tested the possibility that DC stimulation at the surgery site would increase expression of genes related to bone formation relative to expression in autograft alone. Summary of Background Data. DC electrical stimulation as an adjunct treatment in spinal surgery has shown increased fusion rates when compared with autograft alone, yet the biology of such treatment is not fully understood. Methods. Thirty New Zealand White rabbits were entered into the study. A posterolateral, intertransverse process fusion was performed bilaterally at L4–L5, with autogenous bone graft. An implantable DC stimulator was placed across the decorticated transverse processes before placement of autograft. Animals were killed at 3, 7, 14, 21, and 28 days. mRNA levels of BMP-2, 4, 6, 7, VEGF, FGF-2, TGF-β, ALK-2, and ALK-3 were evaluated with real-time RT-PCR. Results. mRNA expression was significantly higher in the DC stimulated animals versus the control animals for several of the genes studied. In particular, levels of mRNA were elevated for BMP-2, BMP-6, and BMP-7. Conclusions. This study shows for the first time that DC stimulation results in a sustained increase of multiple osteogenic genes, suggesting that the biologic mechanism for the DC-induced increase in the rate and extent of bone formation observed clinically may be mediated by the up-regulation of these osteoinductive factors.

B2A Peptide on Ceramic Granules Enhance Posterolateral Spinal Fusion in Rabbits Compared With Autograft

Study Design. Six groups of 10 animals underwent single level, uninstrumented posterolateral lumbar fusions. Graft sites were implanted with autologous bone, autologous bone plus B2A coated granules with concentrations of 0-, 50-, 100-, or 300 &mgr;g/mL, or no graft material. Explanted motion segments were analyzed by Faxitron radiographs, computerized tomography (CT), manual palpation, and histology. Objective. The synthetic peptide B2A2-K-NS (B2A) was evaluated as a novel agent for augmenting spinal fusion in a posterolateral, noninstrumented rabbit lumbar spine fusion model. Summary of Background Data. There have been many efforts to increase the rate of posterolateral spinal fusion while, at the same time minimizing, use of autologous bone with its inherent harvest morbidity. Methods. B2A coated ceramic granules (0-, 50-, 100-, and 300 &mgr;g/mL) were mixed 1:1 with autogenous iliac crest bone and implanted. Autogenous bone alone was used as a positive control. Fusion was assessed at 6 weeks via radiographs and manual palpation. These results were confirmed with histology and CT. Results. Fusion results from palpation and radiographic evaluations were similar. CT and histology confirmed bridging bone across the transverse processes for fused spines. The highest fusion rates were observed in the 100 &mgr;g B2A/mL group—89% in comparison to 33% for uncoated granules (0 &mgr;g B2A/mL) and 63% for autograft alone. These differences were statistically significant. Conclusion. All investigated B2A concentrations demonstrated increased fusion rates. Fusion masses resulting from the implantation of 100 &mgr;g B2A/mL granules demonstrated new woven bone: fused to the transverse processes, within granule pores, bridging bone across the transverse processes, and bridging residual bone graft and granules. It was concluded that each investigated concentration of B2A coated granules in a 1:1 mixture with autograft increased fusion rates in comparison to controls in this rabbit model.

A clinically realistic large animal model of intra-articular fracture that progresses to post-traumatic osteoarthritis

OBJECTIVE Translation of promising treatments for post-traumatic osteoarthritis (PTOA) to patients with intra-articular fracture (IAF) has been limited by the lack of a realistic large animal model. To address this issue we developed a large animal model of IAF in the distal tibia of Yucatan minipigs and documented the natural progression of this injury. DESIGN Twenty-two fractures were treated using open reduction and internal fixation with either an anatomic reduction or an intentional 2-mm step-off. Pre-operatively, and 3 days, 1, 2, 4, 8, and 12 weeks post-operatively, animals were sedated for synovial fluid draws and radiographs. Limb loading was monitored at the same time points using a Tekscan Walkway. Animals were sacrificed at 12 weeks and the limbs were harvested for histological evaluation. RESULTS All animals achieved bony union by 12 weeks, facilitating nearly complete recovery of the initial 60% decrease in limb loading. TNFα, IL1β, IL6, and IL8 concentrations in the fractured limbs were elevated (P < 0.05) at specific times during the 2 weeks after fracture. Histological cartilage degeneration was more severe in the step-off group (0.0001 < P < 0.27 compared to normal) than in the anatomic reconstruction group (0.27 < P < 0.99 compared to normal). CONCLUSIONS This model replicated key features of a human IAF, including surgical stabilization, inflammatory responses, and progression to osteoarthritic cartilage degeneration, thereby providing a potentially useful model for translating promising treatment options to clinical practice.

Effect of Aspirin on Bone Healing in a Rabbit Ulnar Osteotomy Model

BACKGROUND Aspirin is frequently prescribed following orthopaedic surgery. Although there is substantial evidence that some nonsteroidal anti-inflammatory drugs (NSAIDs) are associated with delayed bone healing, there have been few studies of the effects of aspirin on bone healing and, to our knowledge, none on the effects of physiologic dosages. METHODS Following ulnar osteotomy, fifty-six rabbits were administered a placebo (nine rabbits), indomethacin (nine rabbits given 12.5 mg/kg daily), or aspirin at various doses and schedules (2.7 mg/kg daily for ten rabbits, 10 mg/kg daily for nine rabbits, 50 mg/kg twice daily for ten rabbits, and 100 mg/kg three times daily for nine rabbits). The aspirin doses were chosen to span the clinical dosing range. The indomethacin group served as a positive control and as a relative comparison with the effect of aspirin. Radiographs were obtained every two weeks and the animals were killed at eight weeks. Mechanical testing was performed on all rabbits except for six selected for histological evaluation. RESULTS Aspirin delayed bone healing, as demonstrated radiographically and with mechanical testing, in a dose-dependent fashion at salicylate levels equivalent to those resulting from typical human dosing (low-dose aspirin). Receiver operating characteristic analysis demonstrated a plasma salicylate threshold above 20.7 μg/mL predicting delayed bone healing. This approximates a single human dose of 325 mg. Salicylate levels above this threshold were associated with delayed bone healing similar to that caused by indomethacin. Aspirin dosing frequency did not affect bone healing. Mechanical testing was highly predictive of radiographic healing. The interobserver reliability of radiographic assessment of healing at six and eight weeks (kappa = 0.83 and 0.79, respectively) compared favorably with interobserver reliability in previous studies assessing cortical bridging. CONCLUSIONS In a rabbit ulnar osteotomy model, aspirin delayed bone healing with a threshold equivalent to a human dose of 325 mg.

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

Abstract 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. Graphical abstract Figure. No Caption available.

Cellular interactions and bone healing responses to a novel porous tricalcium phosphate bone graft material.

The use of a porous tricalcium phosphate bone void filler (Cellplex TCP, Wright Medical Technology, Inc, Arlington, Tenn) as an alternative to autograft in bone grafting was studied in benchtop, in vitro cell culture, and in vivo preclinical studies. The experimental design included material property quantification, scaffold seeding with mesenchymal stem cells, and implantation in a rabbit segmental defect model. Measured material properties denoted appropriate composition, porosity, and strengths as compared to the literature. Fluid uptake studies and mesenchymal stem cell affinity revealed the scaffolds capabilities as a suitable host for osteoprogenitor cells. In a 1-cm rabbit diaphyseal segmental defect stabilized with an external fixator, tricalcium phosphate was compared to intact autograft, crushed autograft, and open defects. Torsional strengths and stiffnesses of tricalcium phosphate-treated tibia were greater than or equivalent to both intact and crushed autograft controls. Tricalcium phosphate pores exhibited complete bony infiltration histologically. Collectively, the tricalcium phosphate material properties, cell seeding capabilities, and in vivo biological responses give evidence of this implants functionality as a potential alternative for autograft.

Time‐dependent loss of mitochondrial function precedes progressive histologic cartilage degeneration in a rabbit meniscal destabilization model

The goals of this work were to characterize progression of osteoarthritic cartilage degeneration in a rabbit medial meniscus destabilization (MMD) model and then to use the model to identify pre‐histologic disruptions in chondrocyte metabolism under chronically elevated joint contact stresses in vivo. To characterize PTOA progression, 24 rabbits received either MMD or sham surgery. Limb loading was analyzed preoperatively and at regular postoperative intervals using a Tekscan pressure‐sensitive walkway. Animals were euthanized 8 (n = 8 MMD; n = 8 sham) or 26 weeks (n = 8 MMD) postoperatively for histological cartilage evaluation by an objective, semi‐automated Mankin scoring routine. To examine pre‐histologic pathology, MMD was performed on an additional 20 rabbits, euthanized 1 (n = 9) or 4 weeks (n = 10) postoperatively. Chondrocytes were harvested fresh for measurement of mitochondrial function, an intracellular indicator of pathology after mechanical injury. Both MMD and sham surgery caused slight decreases in limb loading which returned to preoperative levels after 2 weeks. Histologically apparent cartilage damage progressed from 8 to 26 weeks after MMD. Changes in chondrocyte respiration were variable at 1 week, but by 4 weeks postoperatively chondrocyte mitochondrial function was significantly reduced. Many human injuries that lead to PTOA are relatively mild, and the cell‐level mechanisms leading to disease remain unclear. We have documented PTOA progression in an animal model of subtle joint injury under continued use, and demonstrated that this model provides a realistic environment for investigation of multi‐stage cellular pathology that develops prior to overt tissue degeneration and which could be targeted for disease modifying treatments.

Assessment of MASTERGRAFT PUTTY as a graft extender in a rabbit posterolateral fusion model.

Study Design. Randomized, controlled study in a laboratory setting. Blinded observations/assessment of study outcomes. Objective. The purpose of this study was to determine the performance characteristics of MASTERGRAFT PUTTY as a bone graft extender in a rabbit posterolateral spine fusion model. Summary of Background Data. The rabbit posterolateral fusion model is an established environment for testing of fusion concepts. It offers the opportunity to obtain radiographical, histological, and biomechanical data on novel fusion materials. Methods. Thirty-six rabbits were entered into the study with 30 used for analysis. Bilateral posterolateral lumbar intertransverse fusions were performed at L5–L6. The lateral two-thirds of the transverse processes were decorticated and covered with graft material: autograft only (2.5–3.0 cc per side), 25% MASTERGRAFT PUTTY/75% autograft (3.0 cc total per side), or 50% MASTERGRAFT PUTTY and 50% autograft (3.0 cc total per side). Animals were humanely killed at 8 weeks postsurgery. Results. The autograft group had a 63% radiographical fusion rate (5 of 8) and correlated with manual palpation results (63%). The 25% MASTERGRAFT PUTTY group had a 73% radiographical fusion rate (8 of 11) and a manual palpation fusion rate of 64%. The 50% MASTERGRAFT PUTTY group demonstrated a 91% (10 of 11) radiographical fusion rate and 73% manual palpation fusion rate. Histologically, no inflammatory reactions were evident regardless of implant. The 2 MASTERGRAFT PUTTY groups had new bone in direct apposition to the MASTERGRAFT ceramic granules. Conclusion. In this commonly used rabbit posterolateral fusion model, MASTERGRAFT PUTTY in an autograft extender mode produces clinically and radiographically similar results to autograft fusion alone.

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

Inhibiting mitochondrial oxidant production after surgical fixation of an intra-articular fracture prevents osteoarthritis in a porcine model. Osteoarthritis—A mitochondrial malady Articular cartilage—the smooth, avascular tissue that covers the bones in joints—can be damaged by traumatic injury, which can lead to osteoarthritis. In response to injury, chondrocytes ramp up mitochondrial activity, producing reactive oxygen species that can cause further tissue damage and cell death. Coleman and colleagues treated intra-articular fractures in a porcine model with an antioxidant or an inhibitor of the mitochondrial electron transport chain. Regulating mitochondrial metabolism prevented osteoarthritis. This work suggests that the mighty mitochondrion is a therapeutic target for 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.