A novel mouse model of hindlimb joint contracture with 3D-printed casts.

Stiff joints formed after trauma, surgery or immobilization are frustrating for surgeons, therapists and patients alike. Unfortunately, the study of contracture is limited by available animal model systems, which focus on the utilization of larger mammals and joint trauma. Here we describe a novel mouse-based model system for the generation of joint contracture using 3D-printed clamshell casts. With this model system we are able to generate both reversible and irreversible contractures of the knee and ankle. Four- or 8-month-old female mice were casted for either 2 or 3 weeks before liberation. All groups formed measurable contractures of the knee and ankle. Younger mice immobilized for less time formed reversible contractures of the knee and ankle. We were able to generate irreversible contracture with either longer immobilization time or the utilization of older mice. The contracture formation translated into differences in gait, which were detectable using the DigiGait® analysis system. This novel model system provides a higher throughput, lower cost and more powerful tool in studying the molecular and cellular mechanisms considering the large existing pool of transgenic/knockout murine strains.

Locally Delivered Ascorbic Acid and ß-Glycerophosphate Augment Local Bone Graft in a Murine Model of 2-Level Posterior Spinal Fusion.

BACKGROUND: Ascorbic acid is involved in collagen biosynthesis and upregulates alkaline phosphatase, potentially alleviating cell senescence and stimulating mesenchymal stem cell proliferation and differentiation into osteoblasts. We hypothesized locally delivered ascorbic acid and ß-glycerophosphate act as a bone graft extender to increase the volume of new bone formed in a murine model of posterior lumbar fusion. METHODS: Collagen sponges were used as delivery vehicles. Sponges were prepared with primary media alone or with the addition of ascorbic acid and ß-glycerophosphate. Fresh morselized bone graft from 12 donor mice was used. Twenty-four healthy male C57BL/6 mice underwent an uninstrumented posterior L3-L5 lumbar fusion. One control group received morselized bone only. A second "sponge control" group received morselized bone with the control collagen sponge. The third group received morselized bone and a collagen sponge with ascorbic acid and ß-glycerophosphate. Three months postoperatively, the lumbar spine underwent high-resolution micro-computed tomography for analysis of bone formation, density, and bridging fusion. RESULTS: Animals receiving ascorbic acid and ß-glycerophosphate had a statistically significant increase in corrected bone volume compared with control and sponge groups, with a 56.3% and 25.4% increase, respectively. Mineralized bone fraction was statistically significantly decreased for animals in the ascorbic acid group compared with control and sponge groups. There was no significant difference in fusion rate between test groups. CONCLUSIONS: Locally delivered ascorbic acid and ß-glycerophosphate in a murine model of posterior spinal fusion yielded statistically significant increases in new bone formation in the lumbar spine but statistically significant decreases in mineralized bone fraction. Differences in fusion rate were not statistically significant. CLINICAL RELEVANCE: This study provides early data suggesting that delivery of ascorbic acid to a spinal fusion site may be beneficial but does not yet establish an indication for clinical use. Further studies are needed to determine optimal dose and delivery of ascorbic acid.

En bloc excision of sacroiliac chondrosarcoma aided by 3D laser printed modeling and stereotactic navigation.

The surgical management of sacro-iliac chondrosarcomas is challenging given their intimate relationship to the nerves and vessels of the pelvis. Osteotomies for en bloc excision can be challenging because of lack of visualization and high risk of injury to pelvic structures. The use of three-dimensional (3D) printed models helps conceptualize the tumor relative to the patient's anatomy. Coupled with stereotactic navigation, safe osteotomy planning and execution can be performed with avoidance of vital nerves and vessels. Very few cases have been reported demonstrating the successful use of these 2 modern technologies for en bloc excision of difficult tumors. We present our technique of using a 3D printed model and navigation for en bloc excision of a large sacro-iliac chondrosarcoma, supplemented with an intraoperative video.

Comparing cost and print time estimates for six commercially-available 3D printers obtained through slicing software for clinically relevant anatomical models.

BACKGROUND: 3D printed patient-specific anatomical models have been applied clinically to orthopaedic care for surgical planning and patient education. The estimated cost and print time per model for 3D printers have not yet been compared with clinically representative models across multiple printing technologies. This study investigates six commercially-available 3D printers: Prusa i3 MK3S, Formlabs Form 2, Formlabs Form 3, LulzBot TAZ 6, Stratasys F370, and Stratasys J750 Digital Anatomy. METHODS: Seven representative orthopaedic standard tessellation models derived from CT scans were imported into the respective slicing software for each 3D printer. For each printer and corresponding print setting, the slicing software provides a print time and material use estimate. Material quantity was used to calculate estimated model cost. Print settings investigated were infill percentage, layer height, and model orientation on the print bed. The slicing software investigated are Cura LulzBot Edition 3.6.20, GrabCAD Print 1.43, PreForm 3.4.6, and PrusaSlicer 2.2.0. RESULTS: The effect of changing infill between 15% and 20% on estimated print time and material use was negligible. Orientation of the model has considerable impact on time and cost with worst-case differences being as much as 39.30% added print time and 34.56% added costs. Averaged across all investigated settings, horizontal model orientation on the print bed minimizes estimated print time for all 3D printers, while vertical model orientation minimizes cost with the exception of Stratasys J750 Digital Anatomy, in which horizontal orientation also minimized cost. Decreasing layer height for all investigated printers increased estimated print time and decreased estimated cost with the exception of Stratasys F370, in which cost increased. The difference in material cost was two orders of magnitude between the least and most-expensive printers. The difference in build rate (cm3/min) was one order of magnitude between the fastest and slowest printers. CONCLUSIONS: All investigated 3D printers in this study have the potential for clinical utility. Print time and print cost are dependent on orientation of anatomy and the printers and settings selected. Cost-effective clinical 3D printing of anatomic models should consider an appropriate printer for the complexity of the anatomy and the experience of the printer technicians.

Identifying a commercially-available 3D printing process that minimizes model distortion after annealing and autoclaving and the effect of steam sterilization on mechanical strength.

BACKGROUND: Fused deposition modeling 3D printing is used in medicine for diverse purposes such as creating patient-specific anatomical models and surgical instruments. For use in the sterile surgical field, it is necessary to understand the mechanical behavior of these prints across 3D printing materials and after autoclaving. It has been previously understood that steam sterilization weakens polylactic acid, however, annealing heat treatment of polylactic acid increases its crystallinity and mechanical strength. We aim to identify an optimal and commercially available 3D printing process that minimizes distortion after annealing and autoclaving and to quantify mechanical strength after these interventions. METHODS: Thirty millimeters cubes with four different infill geometries were 3D printed and subjected to hot water-bath annealing then immediate autoclaving. Seven commercially available 3D printing materials were tested to understand their mechanical behavior after intervention. The dimensions in the X, Y, and Z axes were measured before and after annealing, and again after subsequent autoclaving. Standard and strength-optimized Army-Navy retractor designs were printed using the 3D printing material and infill geometry that deformed the least. These retractors were subjected to annealing and autoclaving interventions and tested for differences in mechanical strength. RESULTS: For both the annealing and subsequent autoclaving intervention, the material and infill geometry that deformed the least, respectively, was Essentium PLA Gray and "grid". Standard retractors without intervention failed at 95 N +/- 2.4 N. Annealed retractors failed at 127.3 N +/- 10 N. Autoclave only retractors failed at 15.7 N +/- 1.4 N. Annealed then autoclaved retractors failed at 19.8 N +/- 3.1 N. Strength-optimized retractors, after the annealing then autoclaving intervention, failed at 164.8 N +/- 12.5 N. CONCLUSION: For 30 mm cubes, the 3D printing material and infill geometry that deformed the least, respectively, was Essentium PLA and "grid". Hot water-bath annealing results in increased 3D printed model strength, however autoclaving 3D prints markedly diminishes strength. Strength-optimized 3D printed PLA Army-Navy retractors overcome the strength limitation due to autoclaving.

3D printed PLA Army-Navy retractors when used as linear retractors yield clinically acceptable tolerances.

BACKGROUND: Modern low-cost 3D printing technologies offer the promise of access to surgical tools in resource scarce areas, however optimal designs for manufacturing have not yet been established. We explore how the optimization of 3D printing parameters when manufacturing polylactic acid filament based Army-Navy retractors vastly increases the strength of retractors, and investigate sources of variability in retractor strength, material cost, printing time, and parameter limitations. METHODS: Standard retractors were printed from various polylactic acid filament spools intra-manufacturer and inter-manufacturer to measure variability in retractor strength. Printing parameters were systematically varied to determine optimum printing parameters. These parameters include retractor width, thickness, infill percentage, infill geometry, perimeter number, and a reinforced joint design. Estimated retractor mass from computer models allows us to estimate material cost. RESULTS: We found statistically significant differences in retractor strength between spools of the same manufacturer and between manufacturers. We determined the true strength optimized retractor to have 30% infill, 3 perimeters, 0.25 in. thickness, 0.75 in. width, and has "Triangle" infill geometry and reinforced joints, failing at more than 15X the threshold for clinically excessive retraction and costs $1.25 USD. CONCLUSIONS: The optimization of 3D printed Army-Navy retractors greatly improve the efficacy of this instrument and expedite the adoption of 3D printing technology in many diverse fields in medicine not necessarily limited to resource poor settings.

Repurposing Human Osteoarthritic Cartilage as a Bone Graft Substitute in an Athymic Rat Posterolateral Spinal Fusion Model.

BACKGROUND: Spinal fusion involves both endochondral and intramembranous bone formation. We previously demonstrated that endochondral cartilage grafts that were derived from human osteoarthritic (OA) articular cartilage can be used as a bone graft in mouse models. We hypothesized that OA cartilage could also be recycled and repurposed as a bone graft substitute in a posterolateral lumbar spinal fusion model in athymic rats. METHODS: OA articular cartilage was obtained from the femoral resection of a healthy 60-year-old man undergoing elective total knee arthroplasty. The chondrocytes recovered from this tissue were dedifferentiated in monolayer tissue culture and then transitioned to culture conditions that promote chondrocyte hypertrophy. The resultant cell pellets were then used as bone graft substitute for single-level posterolateral spinal fusion in 5 athymic rats. Decortication alone was used as the control group. Spinal fusion was assessed with manual palpation, micro-computed tomography, and histologic analysis. RESULTS: In the experimental group, micro-computed tomography at 4 and 8 weeks demonstrated bilateral fusion in 4 of 5 animals and unilateral fusion in 1 animal. At 8 weeks, manual palpation and histologic analysis showed direct correlation with the radiographic findings. Animals undergoing decortication alone failed to generate any spinal fusion. The difference in the fusion rate between groups was statistically significant with respect to both unilateral fusion (P = .047) and bilateral fusion (P = .007). CONCLUSIONS: In the absence of additional surgically implanted bone graft, hypertrophic chondrocyte grafts are sufficient for generating single-level posterolateral lumbar fusion in athymic rats. CLINICAL RELEVANCE: This animal study demonstrates that cartilage harvested from OA knees can be used as a bone graft substitute. Commercially available cell-based bone grafts have previously only used mesenchymal stem cells or osteoblast precursor cells.

The Effects of Topical Vancomycin on Mesenchymal Stem Cells: More May Not Be Better.

BACKGROUND: The use of topical vancomycin is increasingly popular in spine surgery. Large retrospective reviews suggest that topical vancomycin provides a cost-effective decrease in post-operative infection. Currently, there is little that is known about the maximum dose that can be applied locally. When 1 gram of vancomycin is mixed into the bone graft and another 1 gram applied freely in a spine wound, the local concentration of antibiotic ranges from 260-2900 µg/mL in the immediate post-op period and 50-730 µg/mL by the second post-operative day. We hypothesized that exuberant doses of vancomycin would be toxic to mesenchymal stem cells (MSCs). METHODS: Bone marrow was obtained from the femoral canal of patients undergoing routine elective total hip arthroplasty. Mesenchymal stem cells were isolated using plastic adhesion. Cells were exposed to a wide range of doses of vancomycin for 24 hours and then assessed for viability. Osteogenic potential was assessed with alizarin red staining. RESULTS: There was dose-dependent cell death with vancomycin use. MSC death was 9.43% at 400 µg/mL (p=0.047), 13.79% at 1600 µg/mL (p=0.0047), 19.35% at 3200 µg/mL (p<0.0001), 24.82% at 6400 µg/mL (p<0.0001) and 51.83% at 12800 µg/mL of vancomycin (p<0.0001) in comparison to the control group containing no vancomycin. CONCLUSIONS: Our in vitro study suggests that vancomycin has toxic effects on hMSCs, a cell population particularly important for bone formation. In the absence of any clinical evidence suggesting that "more vancomycin is better," and our data suggesting that more vancomycin is harmful in vitro, surgeons electing to use topical vancomycin in spine surgery should restrict their use to the doses currently reported in the available published studies unless specific reasons exist otherwise. This study does not establish a contraindication to the use of topical vancomycin, nor does it suggest that pseudarthroses are attributable to vancomycin use.

Effects of adding epinephrine to arthroscopic irrigation fluid on cultured chondrocyte survival in vitro.

PURPOSE: This study evaluates the effect of low doses of epinephrine contained in common arthroscopic irrigation solutions on viability of in vitro human articular chondrocytes during short-term exposure. METHODS: Isolated cultured human chondrocytes were treated with culture medium, normal saline solution, 1:300,000 epinephrine solution (equivalent to 10 mL of 1:1,000 epinephrine added to a 3-L saline solution bag), or 1:3,000,000 epinephrine solution (equivalent to 1 mL of 1:1,000 epinephrine added to a 3-L saline solution bag) for 1 hour (N = 84). Twenty-four hours after treatment, chondrocyte viability was measured. Statistical analysis was performed with an analysis of variance with Bonferroni post-test. RESULTS: Chondrocyte viability was significantly better when exposed to normal saline solution alone versus high-dose 1:300,000 epinephrine (87.9% ± 5.4% v 74.6% ± 9.4%, P < .05). Exposure to low-dose 1:3,000,000 epinephrine had significantly better survival versus high-dose 1:300,000 epinephrine (85.0% ± 8.3% v 74.6% ± 9.4%, P < .05). There was no difference in viability after exposure to low-dose 1:3,000,000 epinephrine versus normal saline solution (85.0% ± 8.3% v 87.9% ± 5.4%, P > .05). CONCLUSIONS: In vitro, normal saline solution and low-dose 1:3,000,000 epinephrine are significantly less toxic than high-dose 1:300,000 epinephrine to cultured human articular chondrocytes. CLINICAL RELEVANCE: This in vitro study suggests that arthroscopic irrigation fluid containing 1:3,000,000 epinephrine is less chondrotoxic than solutions containing 1:300,000 epinephrine. Surgeons may wish to use the least amount of epinephrine required for adequate visual clarity during surgery. This study does not establish a contraindication to the use of higher doses of epinephrine.

The effect of medial meniscectomy and meniscal allograft transplantation on knee and anterior cruciate ligament biomechanics.

PURPOSE: Our purpose was to evaluate the effect of meniscectomy and meniscal allograft transplant on anterior cruciate ligament (ACL) and knee biomechanics. METHODS: A differential variable reluctance transducer was placed in the ACL of 10 human cadaveric knees to record strain. Tibial displacement from a neutral reference was recorded relative to the position of the femur. Testing was performed at 30 degrees, 60 degrees, and 90 degrees of knee flexion. Six cycles of anterior-posterior loads were applied to the limit of 150 N. After a testing cycle, a medial meniscectomy was performed and the testing cycle was repeated. A meniscal allograft transplant was performed, and a final testing cycle was conducted. ACL strain and tibial displacement in the meniscectomy and meniscal allograft states were compared with the intact-knee state. RESULTS: Tibial displacement after meniscectomy significantly increased at all angles. The meniscal allograft transplant restored tibial displacement to normal values at 30 degrees and 90 degrees. ACL strain increased significantly after meniscectomy at 60 degrees and 90 degrees of flexion, and meniscal allograft transplant returned the strain values to normal at 60 degrees and 90 degrees. CONCLUSIONS: In most cases medial meniscectomy produced a significant increase in tibial displacement relative to the femur, and meniscal allograft transplantation restored displacement values to normal. Meniscectomy increased ACL strain and meniscal allograft transplant restored strain values to normal in 2 of 3 tested flexion angles. CLINICAL RELEVANCE: The absence of the medial meniscus exposes the ACL to increased strain, whereas meniscal allograft lowered the strain on the native ACL. This could have implications for those patients undergoing ACL reconstruction who have concomitant removal of the medial meniscus.

Biomechanics of the anterior longitudinal ligament during 8 g whiplash simulation following single- and contiguous two-level fusion: a finite element study.

STUDY DESIGN: A computational study of anterior longitudinal ligament (ALL) strain in the cervical spine following single- and 2-level fusion during simulated whiplash. OBJECTIVE: To evaluate how cervical fusion alters the peak strain of the ALL in the adjacent motion segments. SUMMARY OF BACKGROUND DATA: Although an in vitro study of ALL strain during whiplash has been conducted in healthy cervical spines, no such study has been performed in a cervical spine with fused segments. It has been demonstrated that the loss of motion following fusion results in increased strain in the adjacent motion segments. However, the biomechanics of the adjacent motion segments during high energy acceleration-deceleration simulations have not been widely reported. Accordingly, we investigated the peak strain of the ALL following single- and 2-level fusion during simulated whiplash. METHODS: A detailed finite element (FE) model of the human body in the driver-occupant position was used to investigate cervical hyperextension injury. The cervical spine was subjected to simulated whiplash at 8 g acceleration and peak ALL strains were computed. The results were validated against published experimental data. This validated FE model was then modified to simulate single- and 2-level fusion and tested under identical loading conditions. RESULTS: The mean increase in peak ALL strain at the motion segment immediately adjacent to the level of fusion was 15.5% for single-level fusion when compared with 40.8% in 2-level contiguous fusion (P = 0.019). CONCLUSION: Cervical arthrodesis increases peak ALL strain in the adjacent motion segments. Two-level fusion increased ALL strain in the adjacent motion segments, on average, greater than single-level fusion did. Disc arthroplasty and other techniques that provide stability without loss of flexibility may be beneficial in patients undergoing multiple-level fusion. Detailed FE models such as ours can provide strong correlation with experimentally determined data.

Effect of ventricular size and patch stiffness in surgical anterior ventricular restoration: a finite element model study.

BACKGROUND: Surgical anterior ventricular restoration (SAVER) has been proposed for dilated ischemic cardiomyopathy with an akinetic distal anterior left ventricular wall. We tested the hypothesis that SAVER increases stroke volume, reduces mean myofiber stress and achieves optimal results without a patch. METHODS: A finite element model of the left ventricle (LV) with an akinetic but contractile anteroapical LV wall segment was used. Separate versions of the model with normal and dilated LV sizes were developed and used to simulate the SAVER operation with and without a patch of varying stiffness from 10 to 100 kilopascals. RESULTS: The SAVER operation reduced myofiber stress in the akinetic infarct and infarct borderzone, but caused a reduction in the Starling relationship. In all cases, stroke volume decreased while ejection fraction increased after SAVER. The SAVER operation was more beneficial in dilated ventricles, and the reduction in stroke volume after SAVER without patch was minimal. The effect of patch stiffness was mixed as stiffer material causes a greater reduction in stress yet has the greatest negative effect on stroke volume. CONCLUSIONS: These simulations support the use of SAVER in dilated hearts without a patch.

Akinetic myocardial infarcts must contain contracting myocytes: finite-element model study.

Infarcted segments of myocardium demonstrate functional impairment ranging in severity from hypokinesis to dyskinesis. We sought to better define the contributions of passive material properties (stiffness) and active properties (contracting myocytes) to infarct thickening. Using a finite-element (FE) model, we tested the hypothesis that infarcted myocardium must contain contracting myocytes to be akinetic and not dyskinetic. A three-dimensional FE mesh of the left ventricle was developed with echocardiographs from a reperfused ovine anteroapical infarct. The nonlinear stress-strain relationship for the diastolic myocardium was anisotropic with respect to the local muscle fiber direction, and an elastance model for active fiber stress was incorporated. The diastolic stiffness (C) and systolic material property (isometric tension at longest sarcomere length and peak intracellular calcium concentration, T(max)) of the uninfarcted remote myocardium were assumed to be normal (C = 0.876 kPa, T(max) = 135.7 kPa). Diastolic and systolic properties of the infarct necessary to produce akinesis, defined as an average radial strain between -0.01 and 0.01, were determined by assigning a range of diastolic stiffnesses and scaling infarct T(max) to represent the percentage of contracting myocytes between 0% and 100%. As C was increased to 11 times normal (C = 10 kPa) the percentage of T(max) necessary for akinesis increased from 20% to 50%. Without contracting myocytes, C = 250 kPa was necessary to achieve akinesis. If infarct stiffness is <285 times normal, contracting myocytes are required to prevent dyskinetic infarct wall motion.