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.

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.

Mechanosensitive Control of Articular Cartilage and Subchondral Bone Homeostasis in Mice Requires Osteocytic Transforming Growth Factor ß Signaling.

OBJECTIVE: Transforming growth factor ß (TGFß) signaling plays a complex tissue-specific and nonlinear role in osteoarthritis (OA). This study was conducted to determine the osteocytic contributions of TGFß signaling to OA. METHODS: To identify the role of osteocytic TGFß signaling in joint homeostasis, we used 16-week-old male mice (n = 9-11 per group) and female mice (n = 7-11 per group) with an osteocyte-intrinsic ablation of TGFß receptor type II (TßRIIocy-/- mice) and assessed defects in cartilage degeneration, subchondral bone plate (SBP) thickness, and SBP sclerostin expression. To further investigate these mechanisms in 16-week-old male mice, we perturbed joint homeostasis by subjecting 8-week-old mice to medial meniscal/ligamentous injury (MLI), which preferentially disrupts the mechanical environment of the medial joint to induce OA. RESULTS: In all contexts, independent of sex, genotype, or medial or lateral joint compartment, increased SBP thickness and SBP sclerostin expression were spatially associated with cartilage degeneration. Male TßRIIocy-/- mice, but not female TßRIIocy-/- mice, had increased cartilage degeneration, increased SBP thickness, and higher levels of SBP sclerostin compared with control mice (all P < 0.05), demonstrating that the role of osteocytic TGFß signaling on joint homeostasis is sexually dimorphic. With changes in joint mechanics following injury, control mice had increased SBP thickness, subchondral bone volume, and SBP sclerostin expression (all P < 0.05). TßRIIocy-/- mice, however, were insensitive to subchondral bone changes with injury, suggesting that mechanosensation at the SBP requires osteocytic TGFß signaling. CONCLUSION: Our results provide new evidence that osteocytic TGFß signaling is required for a mechanosensitive response to injury, and that osteocytes control SBP homeostasis to maintain cartilage health, identifying osteocytic TGFß signaling as a novel therapeutic target for OA.

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.

Osteocyte dysfunction promotes osteoarthritis through MMP13-dependent suppression of subchondral bone homeostasis.

Osteoarthritis (OA), long considered a primary disorder of articular cartilage, is commonly associated with subchondral bone sclerosis. However, the cellular mechanisms responsible for changes to subchondral bone in OA, and the extent to which these changes are drivers of or a secondary reaction to cartilage degeneration, remain unclear. In knee joints from human patients with end-stage OA, we found evidence of profound defects in osteocyte function. Suppression of osteocyte perilacunar/canalicular remodeling (PLR) was most severe in the medial compartment of OA subchondral bone, with lower protease expression, diminished canalicular networks, and disorganized and hypermineralized extracellular matrix. As a step toward evaluating the causality of PLR suppression in OA, we ablated the PLR enzyme MMP13 in osteocytes while leaving chondrocytic MMP13 intact, using Cre recombinase driven by the 9.6-kb DMP1 promoter. Not only did osteocytic MMP13 deficiency suppress PLR in cortical and subchondral bone, but it also compromised cartilage. Even in the absence of injury, osteocytic MMP13 deficiency was sufficient to reduce cartilage proteoglycan content, change chondrocyte production of collagen II, aggrecan, and MMP13, and increase the incidence of cartilage lesions, consistent with early OA. Thus, in humans and mice, defects in PLR coincide with cartilage defects. Osteocyte-derived MMP13 emerges as a critical regulator of cartilage homeostasis, likely via its effects on PLR. Together, these findings implicate osteocytes in bone-cartilage crosstalk in the joint and suggest a causal role for suppressed perilacunar/canalicular remodeling in osteoarthritis.

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.

Clavicle Fracture Malunion Treated with an Osteotomy Guided by a Three-Dimensional-Printed Model: A Case Report.

CASE: This case report describes the management of a chronic and symptomatic clavicle malunion with use of a 3-dimensional (3D)-printed model during the preoperative surgical planning. CONCLUSION: The use of 3D printing has many applications in the medical field. Constant improvement in the quality of 3D printing has contributed to its increased use in a variety of surgeries. In our patient, 3D printing was used to generate a surface model of the clavicle fracture malunion and the "mirrored" contralateral healthy clavicle to plan an intraoperative osteotomy, which optimized the relative position of the osteotomy segments and hardware fixation.

Rotator Cuff Disease: Treatment Options and Considerations.

Rotator cuff disease encompasses a broad spectrum of injury and pathology with an increasing incidence with age. Pain with overhead activity, localizing to the deltoid region, and loss of active range of motion of the shoulder are among the most common presenting symptoms. Treatment options are dependent on the extent of disease and patient symptoms, and may range from physical therapy to surgical repair using a variety of possible techniques. Tear thickness, size, and morphology frequently dictate the repair techniques that are used, such as margin convergence, anterior and posterior interval slides, and mobilization of the rotator interval and supraspinatus with a subscapularis repair. Establishing and maintaining a low-tension repair is important in optimizing tendon healing after surgery. Superior capsule reconstruction is an emerging treatment strategy for massive, otherwise irreparable cuff tears, though more long-term evidence is necessary to fully evaluate this option.

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.

Kartogenin treatment prevented joint degeneration in a rodent model of osteoarthritis: A pilot study.

Osteoarthritis (OA) is a major degenerative joint disease characterized by progressive loss of articular cartilage, synovitis, subchondral bone changes, and osteophyte formation. Currently there is no treatment for OA except temporary pain relief and end-stage joint replacement surgery. We performed a pilot study to determine the effect of kartogenin (KGN, a small molecule) on both cartilage and subchondral bone in a rat model of OA using multimodal imaging techniques. OA was induced in rats (OA and KGN treatment group) by anterior cruciate ligament transection (ACLT) surgery in the right knee joint. Sham surgery was performed on the right knee joint of control group rats. KGN group rats received weekly intra-articular injection of 125 µM KGN 1 week after surgery until week 12. All rats underwent in vivo magnetic resonance imaging (MRI) at 3, 6, and 12 weeks after surgery. Quantitative MR relaxation measures (T1ρ and T2 ) were determined to evaluate changes in articular cartilage. Cartilage and bone turnover markers (COMP and CTX-I) were determined at baseline, 3, 6, and 12 weeks. Animals were sacrificed at week 12 and the knee joints were removed for micro-computed tomography (micro-CT) and histology. KGN treatment significantly lowered the T1ρ and T2 relaxation times indicating decreased cartilage degradation. KGN treatment significantly decreased COMP and CTX-I levels indicating decreased cartilage and bone turnover rate. KGN treatment also prevented subchondral bone changes in the ACLT rat model of OA. Thus, kartogenin is a potential drug to prevent joint deterioration in post-traumatic OA. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1780-1789, 2016.

Novel mouse model of spinal cord injury-induced heterotopic ossification.

Heterotopic ossification (HO) develops in about 20% to 30% of patients with spinal cord injury (SCI) and significantly impairs their rehabilitation. There is no effective prevention or treatment for this condition at this time. Our current understanding of its etiology and pathophysiology is limited partially due to the lack of clinically relevant animal models. In this study, we report a novel mouse model of SCI-induced HO by administering a subthreshold dose of bone morphogenetic protein (BMP)-2 to muscles in mice after SCI. Micro-computed tomography scanning showed that an intramuscular injection of 0.25 micrograms of BMP-2 causes significant HO in mice with SCI but not in control (sham surgery) mice. Our analysis of gene expression showed significantly increased BMP signaling in quadriceps following SCI, suggesting that BMP signaling may play a role in SCI-induced HO. Administering 0.25 micrograms of BMP-2 to the front arms of the mice with SCI also results in the development of significant HO but not in control mice. This suggests that SCI causes a systematic osteogenic effect, which is not limited to paralyzed limbs. This novel mouse model will serve as a powerful tool in exploring the molecular mechanisms of SCI-induced HO, which may lead to novel treatment for this disease.

Reduction of environmental temperature mitigates local anesthetic cytotoxicity in bovine articular chondrocytes.

The purpose of this study was to assess whether reducing environmental temperature will lead to increased chondrocyte viability following injury from a single-dose of local anesthetic treatment. Bovine articular chondrocytes from weight bearing portions of femoral condyles were harvested and cultured. 96-well plates were seeded with 15,000 chondrocytes per well. Chondrocytes were treated with one of the following conditions: ITS Media, 1x PBS, 2% lidocaine, 0.5% bupivacaine, or 0.5% ropivacaine. Each plate was then incubated at 37°C, 23°C, or 4°C for one hour and then returned to media at 37°C. Chondrocyte viability was assessed 24 hours after treatment. Chondrocyte viability is presented as a ratio of the fluorescence of the treatment group over the average of the media group at that temperature (ratio ± SEM). At 37°C, lidocaine (0.35 ± 0.04) and bupivacaine (0.30 ± 0.05) treated chondrocytes show low cell viability when compared to the media (1.00 ± 0.03) control group (p < 0.001). Lidocaine treated chondrocytes were significantly more viable at 23°C (0.84 ± 0.08) and 4°C (0.86±0.085) than at 37°C (p < 0.001). Bupivacaine treated chondrocytes were significantly more viable at 4°C (0.660 ± 0.073) than at 37°C or 23°C (0.330 ± 0.069) (p < 0.001 and p = 0.002 respectively). Reducing the temperature from 37°C to 23°C during treatment with lidocaine increases chondrocyte viability following injury. Chondrocytes treated with bupivacaine can be rescued by reducing the temperature to 4°C. Key pointsConfirm that local anesthetics, specifically bupivacaine and lidocaine, are toxic to chondrocytes in monolayerChondrocyte viability significantly improved for chondrocytes treated with bupivacaine when the environment was cooled to 23°C.Chondrocyte viability significantly improved for chondrocytes treated with bupivacaine or lidocaine when the environment was cooled to 4°CIt is the recommendation of the authors that physicians should be wary of the risks of injecting local anesthetics into the intra-articular space.Active cooling of the joint could potentially protect the articular cartilage from insult following treatment with local anesthetics.

Joint instability and cartilage compression in a mouse model of posttraumatic osteoarthritis.

Joint instability and cartilage trauma have been previously studied and identified as key mediators in the development of posttraumatic osteoarthritis (PTOA). The purpose of this study was to use an in vivo model to compare the effect of joint instability, caused by the rupture of the anterior cruciate ligament (ACL), versus cartilage compression. In this study, mice were subjected to cyclical axial loads of twelve Newtons (N) for 240 cycles or until the ACL ruptured. One and eight weeks after this procedure, knees were sectioned coronally and evaluated for osteoarthritis by histology. Using a scoring scale established by [Pritzker K, Gay S, Jimenez S, et al. (2006): Osteoarthritis Cartilage 14:13-29], the articular cartilage across each surface was scored and combined to produce a total degeneration score. The ACL-ruptured group had a significantly greater total degeneration score than either control or compression treated joints at 1 and 8 weeks. Additionally, only sections from ACL-ruptured knees consistently showed synovitis after 1 week and osteophyte formation after 8 weeks. Thus, it appears using that ACL rupture consistently creates a severe osteoarthritis phenotype, while axial cartilage compression alone does not appear to be an appropriate method of inducing PTOA in vivo.

Cytotoxicity of local anesthetics on human mesenchymal stem cells.

BACKGROUND: Local anesthetics are frequently delivered intra-articularly to provide perioperative pain control. Previous studies have shown that the commonly used drugs lidocaine, ropivacaine, and bupivacaine can be toxic to human chondrocytes. The present study was conducted to determine whether the toxic effects of local anesthetics on human chondrocytes also extend to human mesenchymal stem cells. METHODS: Human mesenchymal stem cells from three healthy donors were grown in tissue culture and exposed to the following anesthetic treatments for sixty minutes: (1) 1% lidocaine, (2) 2% lidocaine, (3) 0.25% bupivacaine, (4) 0.5% bupivacaine, (5) 0.2% ropivacaine, and (6) 0.5% ropivacaine. The cells were then allowed to recover for twenty-four hours in regular growth media, and viability was measured with use of fluorescent staining for live cells or a luminescence assay for ATP content. RESULTS: The live cell counts and ATP content were correlated (r2 = 0.79), and 2% lidocaine was found to be significantly more toxic than all doses of bupivacaine and ropivacaine. Treatment with 1% lidocaine resulted in significantly fewer live cells (49%) compared with the control, and the live cell count was also significantly less than that for the other anesthetics. However, the ATP level in the 1% lidocaine group was not significantly lower than those in the other groups. Bupivacaine and ropivacaine did not exhibit significant differences in toxicity compared with the control or with each other. CONCLUSIONS: Ropivacaine and bupivacaine had limited toxicity in human mesenchymal stem cells. However, lidocaine could significantly decrease mesenchymal stem cell viability. Since other studies have shown ropivacaine to be less toxic to chondrocytes than bupivacaine, ropivacaine may be a safer intra-articular anesthetic. CLINICAL RELEVANCE: Mesenchymal stem cells likely play a key role in healing following surgical procedures such as microfracture and ligament reconstruction. If local anesthetics are used following joint surgery, selection of an agent with low toxicity toward mesenchymal stem cells, such as ropivacaine, may maximize tissue healing potential.

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.

Chondrocyte apoptosis after simulated intraarticular fracture: a comparison of histologic detection methods.

Accurate evaluation of programmed cell death, or apoptosis, in chondrocytes is essential to studying cartilage injury. We evaluated four methods of detecting chondrocyte-programmed cell death in formalin-fixed, paraffin-embedded cartilage after experimental osteochondral fracture. Human osteochondral explants were subjected to experimental fracture in a manner known to induce high levels of chondrocyte-programmed cell death. After 4 days in culture, specimens were fixed and analyzed for programmed cell death using: (1) terminal deoxynucleotidyl transferase end labeling; (2) DNA denaturation analysis using an antibody specific for single-stranded DNA; (3) immunohistochemistry using antisera specific for active caspase-3; and (4) in situ oligonucleotide ligation. Quantitative analysis of programmed cell death levels for each technique was performed comparing injured and uninjured areas of cartilage. We observed differences between injured and uninjured areas of cartilage using the four methods. Human cartilage fixed in zinc-formalin and embedded in paraffin is amenable to programmed cell death analysis using any of four independent methods, each of which ostensibly has some advantages in terms of assaying different steps along the apoptotic pathway. Using the protocols described in this article, investigators may have additional tools to identify and quantify chondrocytes undergoing programmed cell death after experimental cartilage injury.

Inhibition of MMP2/MMP9 after spinal cord trauma reduces apoptosis.

STUDY DESIGN: Randomized controlled trial. OBJECTIVE: To characterize the increase in gelatinase A (MMP2) activity after spinal cord injury (SCI) in the mouse model, and the effects of MMP2/MMP9 inhibition on apoptotic cells. SUMMARY OF BACKGROUND DATA: Clinical consequences of SCI are due to a series of secondary injury cascades. Matrix metalloproteinases are thought play a key role in this, leading to apoptotic cell death. METHODS: SCI via a drop tower in mice was used. MMP2 beta-gal reporter mice were used to quantify the level of MMP2 after SCI. In a follow-up experiment, mice which underwent SCI were randomized to daily SQ injections of MMP2/MMP9 inhibitor versus placebo. MMP2 levels were quantified and histology was performed with TUNEL and Luxol fast blue staining. RESULTS: MMP2 transcription was significantly upregulated after SCI, by the beta-gal assay. Inhibition of MMP2/MMP9 activity after SCI led to statistically significant decreases in apoptosis within the zone of injury. There was a trend towards preservation of myelin by preserved luxol fast blue staining. CONCLUSION: After SCI, MMP2 is upregulated along with neuron and glial cells apoptosis. The level of apoptosis could be reduced with MMP2/MMP9 inhibition. This supports MMP2 as cause for apoptosis after SCI with the potential for therapeutic intervention as apoptosis can be reduced with MMP2 inhibition.

Chondrocyte apoptosis: implications for osteochondral allograft transplantation.

Osteochondral allograft transplantation is a useful technique to manage larger articular cartilage injuries. One factor that may compromise the effectiveness of this procedure is chondrocyte cell death that occurs during the storage, preparation, and implantation of the osteochondral grafts. Loss of viable chondrocytes may negatively affect osteochondral edge integration and long-term function. A better understanding of the mechanisms responsible for chondrocyte loss could lead to interventions designed to decrease cell death and improve results. Recent studies indicate that apoptosis, or programmed cell death, is responsible for much of the chondrocyte death associated with osteochondral allograft transplantation. Theoretically, some of these cells can be rescued by blocking important apoptotic mediators. We review the role of apoptosis in cartilage degeneration, focusing on apoptosis associated with osteochondral transplantation. We also review the pathways thought to be responsible for regulating chondrocyte apoptosis, as well as experiments testing inhibitors of the apoptotic pathway. These data suggest that key contributors to the apoptotic process can be manipulated to enhance chondrocyte survival. This knowledge may lead to better surgical outcomes for osteochondral transplantation.

Hemodynamic and oxygen transport patterns after head trauma and brain death: implications for management of the organ donor.

OBJECTIVES: The aims of the present study were to describe the temporal hemodynamic and oxygen transport patterns of patients with head injuries as well as the patterns of those who became brain dead to better understand the role of underlying central regulatory hemodynamic mechanisms and ultimately to improve rates of organ donation. METHODS: We studied 388 consecutive noninvasively monitored patients with severe head trauma; 79 of these became brain dead. Monitoring was started shortly after admission to the emergency department and was designed to describe the sequence of cardiac, pulmonary, and tissue perfusion functions by cardiac index (CI), mean arterial pressure, heart rate, arterial saturation by pulse oximetry (Sapo2), and transcutaneous oxygen and carbon dioxide (Ptco2/Fio2 and Ptcco2) patterns. The latter were used as markers of tissue perfusion or oxygenation. RESULTS: Patients with head injuries who subsequently became brain dead initially had low CI with poor tissue perfusion beginning shortly after emergency department admission. This was followed by a prolonged period characterized by high CI (4.43 +/- 1.3 L x min(-1) x m2) and enhanced tissue oxygenation (Ptco2/Fio2 238 +/- 186). In the late or end stage of brain death, hemodynamic deterioration and collapse led rapidly to arrest. In attempts to maintain hemodynamic stability for organ donation, the effects of various therapies on the hemodynamic patterns were preliminarily described. CONCLUSIONS: The hyperdynamic state with exaggerated peripheral tissue perfusion or oxygenation in brain-dead patients associated with loss of central vasoconstrictive mechanisms of the stress response resulted in unopposed peripheral metabolic vasodilatation producing high CI and tissue perfusion.