Alexis C. Dang

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.

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 &bgr;-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 &bgr;-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.

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.

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.

Inhibition of chondrocyte apoptosis in vivo following acute osteochondral injury

In spite of advances and refinements in surgical technique, the development of post-traumatic arthritis following osteochondral injury remains one of the major unsolved problems faced by orthopedic surgeons. Of particular interest is the possibility that chondrocyte programmed cell death (PCD), or ‘apoptosis’, contributes to the subsequent development of post-traumatic arthritis 1–5 . Recent studies have shown that inhibitors of caspases, key enzymes in the apoptosis pathway, can block chondrocyte PCD in vitro 4–7 . The goal of the presented study was to test the hypothesis that short-term, intra-articular caspase inhibitor treatment can limit chondrocyte PCD in vivo following acute osteochondral injury. Adult New Zealand white rabbits underwent experimental osteochondral injury. One group of rabbits received daily intra-articular injections of the broadspectrum caspase inhibitor Z-VAD-fmk. A control group of rabbits received daily intra-articular injections of vehicle alone. After four days of treatment, the articular cartilage was analyzed for chondrocyte PCD. Treatment with Z-VADfmk resulted in a marked reduction in the percentage of chondrocytes undergoing PCD compared to controls [treated=7.4±1.8%; controls=32.5±8.6% (P<0.0001 by student’s t-test)]. The degree of PCD inhibition was dependent upon the distance from the site of injury, with levels of PCD reduced to background levels in areas beyond 1 mm from the drill hole. These results suggest that the intraarticular administration of PCD inhibitors may be a useful strategy for the treatment of osteochondral injury by limiting the extent of articular chondrocyte loss due to apoptosis. For these experiments, a drill with a cooled 2 mm bit was used to create osteochondral injuries in the hind limb femoral condyles of adult rabbits. The injuries were created in the weight bearing portions of the condyles and penetrated into the subchondral bone. Following surgery, five animals received daily intra-articular injections of the caspase inhibitor Z-VAD-fmk (100 µM in 0.1% DMSO, 0.5 cc; Calbiochem, CA) delivered via an indwelling catheter. A control group of five rabbits received daily intra-articular injections of vehicle alone. The knee tissue was harvested on post-injury day 4, decalcified in EDTA, embedded in paraffin, and then sectioned in the sagittal plane at 5 µm thickness for further analysis. Chondrocyte apoptosis was quantified by TUNEL analysis using the ApopTag ® Direct in situ apoptosis detection kit (Intergen, NY) with DAPI used as a counterstain. Fluorescence images were captured using appropriate filters with an Axiocam digital camera (Zeiss, NJ) at 1 megapixel resolution. Each captured field was subdivided into three ‘Zones’ with Zone 1 encompassing the full thickness of articular cartilage extending from 0 to 0.5 mm away from the drill hole; Zone 2 extending from 0.5 to 1 mm away from the drill hole; and Zone 3 extending from 1 to 1.5 mm away from the drill hole (Fig. 1). Semi-automated data collection and analysis were performed using custom

Characterisation of osteoarthritis in a small animal model of type 2 diabetes mellitus

Objectives Our objective in this article is to test the hypothesis that type 2 diabetes mellitus (T2DM) is a factor in the onset and progression of osteoarthritis, and to characterise the quality of the articular cartilage in an appropriate rat model. Methods T2DM rats were obtained from the UC Davis group and compared with control Lewis rats. The diabetic rats were sacrificed at ages from six to 12 months, while control rats were sacrificed at six months only. Osteoarthritis severity was determined via histology in four knee quadrants using the OARSI scoring guide. Immunohistochemical staining was also performed as a secondary form of osteoarthritic analysis. Results T2DM rats had higher mean osteoarthritis scores than the control rats in each of the four areas that were analysed. However, only the results at the medial and lateral femur and medial tibia were significant. Cysts were also found in T2DM rats at the junction of the articular cartilage and subchondral bone. Immunohistochemical analysis does not show an increase in collagen II between control and T2DM rats. Mass comparisons also showed a significant relationship between mass and osteoarthritis score. Conclusions T2DM was found to cause global degeneration in the UCD rat knee joints, suggesting that diabetes itself is a factor in the onset and progression of osteoarthritis. The immunohistochemistry stains showed little to no change in collagen II degeneration between T2DM and control rats. Overall, it seems that the animal model used is pertinent to future studies of T2DM in the development and progression of osteoarthritis. Cite this article: Bone Joint Res 2014;3:203–11

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.

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.

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.