Chondrocyte Culture Parameters for Matrix-Assisted Autologous Chondrocyte Implantation Affect Catabolism and Inflammation in a Rabbit Model.

Cartilage defects represent an increasing pathology among active individuals that affects the ability to contribute to sports and daily life. Cell therapy, such as autologous chondrocyte implantation (ACI), is a widespread option to treat larger cartilage defects still lacking standardization of in vitro cell culture parameters. We hypothesize that mRNA expression of cytokines and proteases before and after ACI is influenced by in vitro parameters: cell-passage, cell-density and membrane-holding time. Knee joint articular chondrocytes, harvested from rabbits (n = 60), were cultured/processed under varying conditions: after three different cell-passages (P1, P3, and P5), cells were seeded on 3D collagen matrices (approximately 25 mm³) at three different densities (2 × 105/matrix, 1 × 106/matrix, and 3 × 106/matrix) combined with two different membrane-holding times (5 h and two weeks) prior autologous transplantation. Those combinations resulted in 18 different in vivo experimental groups. Two defects/knee/animal were created in the trochlear groove (defect dimension: ∅ 4 mm × 2 mm). Four identical cell-seeded matrices (CSM) were assembled and grouped in two pairs: One pair giving pre-operative in vitro data (CSM-i), the other pair was implanted in vivo and harvested 12 weeks post-implantation (CSM-e). CSMs were analyzed for TNF-α, IL-1ß, MMP-1, and MMP-3 via qPCR. CSM-i showed higher expression of IL-1ß, MMP-1, and MMP-3 compared to CSM-e. TNF-α expression was higher in CSM-e. Linearity between CSM-i and CSM-e values was found, except for TNF-α. IL-1ß expression was higher in CSM-i at higher passage and longer membrane-holding time. IL-1ß expression decreased with prolonged membrane-holding time in CSM-e. For TNF-α, the reverse was true. Lower cell-passages and lower membrane-holding time resulted in stronger TNF-α expression. Prolonged membrane-holding time resulted in increased MMP levels among CSM-i and CSM-e. Cellular density was of no significant effect. We demonstrated cytokine and MMP expression levels to be directly influenced by in vitro culture settings in ACI. Linearity of expression-patterns between CSM-i and CSM-e may predict ACI regeneration outcome in vivo. Cytokine/protease interaction within the regenerate tissue could be guided via adjusting in vitro culture parameters, of which membrane-holding time resulted the most relevant one.

Operative R0 resection of diffuse large B-cell lymphoma of the pelvis: a case report.

BACKGROUND: Diffuse large B-cell lymphoma is the most common subtype of non-Hodgkin lymphoma with or without involvement of extranodal sites. Rituximab in combination with cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP) therapy represents the current standard therapy, achieving a rather dissatisfying outcome in approximately 30-40% of all cases. CASE PRESENTATION: We present the case of a 43-year-old Austrian woman with an incidentally detected large pelvic mass which was diagnosed as diffuse large B-cell lymphoma. Initially, the lymphoma intraoperatively appeared to be an inoperable conglomerate tumor. Soon, intestinal perforation induced by tumor infiltration occurred, which initiated a closure of the small intestine and application of a jejunal probe and a percutaneous endoscopic gastrotomy tube. Treatment utilizing the gold standard rituximab in combination with cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP) was performed, partly resulting in remission according to radiological follow-up. In view of diagnosis and primary treatment development, the predictive outcome appeared unsound. However, within the procedure of the latest surgical intervention, which was intended to at least reconstruct the intestinal passage in order to improve quality of life, a surgical R0 resection of the residual tumor mass was achieved. CONCLUSIONS: The case presented here reports an unanticipated process of diffuse large B-cell lymphoma, underlining the importance of interdisciplinary cooperation and surgical intervention within the realms of state-of-the-art treatment.

Effect of COX-2 inhibition on tendon-to-bone healing and PGE2 concentration after anterior cruciate ligament reconstruction.

BACKGROUND: Non-steroidal anti-inflammatory drugs are commonly used to reduce pain and inflammation in orthopaedic patients. Selective cyclooxygenase-2 (COX-2) inhibitors have been developed to minimize drug-specific side effects. However, they are suspected to impair both bone and tendon healing. The objective of this study is to evaluate the effect of COX-2 inhibitor administration on tendon-to-bone healing and prostaglandin E (PGE2) concentration. METHODS: Thirty-two New Zealand white rabbits underwent reconstructions of the anterior cruciate ligaments and were randomized into four groups: Two groups postoperatively received a selective COX-2 inhibitor (Celecoxib) on a daily basis for 3 weeks, the two other groups received no postoperative COX-2 inhibitors at all and were examined after three or 6 weeks. The PGE2 concentration of the synovial fluid, the osseous integration of the tendon graft at tunnel aperture and midtunnel section, as well as the stability of the tendon graft were examined via biomechanic testing. RESULTS: After 3 weeks, the PGE2 content of the synovial fluid in the COX-2 inhibitor recipients was significantly lower than that of the control group (p = 0.018). At the same time, the COX-2 inhibitor recipients had a significantly lower bone density and lower amount of new bone formation than the control group (p = 0.020; p = 0.028) in the tunnel aperture. At the 6-week examination, there was a significant increase in the PGE2 content within synovial fluid of the COX-2 inhibitor recipients (p = 0.022), whose treatment with COX-2 inhibitors had ended 3 weeks earlier; in contrast, the transplant stability decreased and was reduced by 37% compared to the controls. CONCLUSIONS: Selective COX-2 inhibitors cause impaired tendon-to-bone healing, weaken mechanical stability and decrease PGE2 content of the synovial fluid. The present study suggests a reluctant use of COX-2 inhibitors when tendon-to-bone healing is intended.

Longitudinal changes in subchondral bone structure as assessed with MRI are associated with functional outcome after high tibial osteotomy.

OBJECTIVES: The primary objective of this study was to evaluate the effects of high tibial osteotomy (HTO) on subchondral bone structure assessed with magnetic resonance (MR)-based trabecular bone imaging and the correlations of these effects with functional outcome and clinical symptoms. METHODS: Patients with varus malalignment (6.2±2.2°) and without a history of knee surgery (n=22; 3 women; 48.7±10.3 years) were included into this prospective study. 1.5T MR imaging was performed before and on average 1.5 years after HTO (amount of correction 4.7±2.5°) and histomorphometric parameters of the trabecular bone were calculated for the medial/ lateral tibia and femur. Functional outcome was assessed with validated scores focusing on sports activity including the Lysholm Score, Tegner Activity Scale and the adapted Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) Score. RESULTS: Apparent trabecular number significantly decreased in all compartments of the tibiofemoral joint when comparing values before and on average 1.5 years after HTO (P<0.05 for all). Decrease in apparent trabecular number was significantly higher within the medial tibia compared to the lateral compartment (mean difference -0.24 mm-1 (95% confidence interval (CI): -0.33, -0.14 mm-1); P<0.001). Apparent trabecular bone thickness significantly increased within 1.5 years after HTO in the lateral femur (P=0.002) and tibia (P <0.001). The Lysholm Score and Tegner Scale demonstrated an improvement of functional outcome, and the adapted WOMAC demonstrated an improvement of pain, stiffness and physical function within 1.5 years after HTO (P<0.01), with the improvement of WOMAC correlating significantly with changes in trabecular bone thickness within the medial tibia (r= -0.48; P=0.01). CONCLUSION: These findings indicate a reversal of the previous subchondral bone alterations in patients with varus malalignment after undergoing HTO, while pronounced subchondral changes were associated with a better functional outcome. LEVEL OF EVIDENCE: 3.

Analysis of the effects of high tibial osteotomy on tibial rotation.

PURPOSE: Limited literature reports on internal and external rotation of the distal fragment in the context of valgus open wedge (OW) high tibial osteotomy (HTO). In the authors clinical observation, the distal fragment was always rotated internally in relation to the proximal fragment by the end of the surgical procedure. The purpose was to evaluate the influence of valgus OW-HTO on post-operative tibial torsion. STUDY DESIGN: Prospective case series. METHODS: Fifty patients (10 female, 40 male; mean age 42.1 ± 9.4 years) underwent valgus OW- HTO. The osteotomy was spread and fixed with a locking plate at the posteromedial aspect of the proximal tibia. The osteotomy of the tibial tuberosity was performed either proximally or distally dependent on the patello-femoral findings. Two independent observers measured axial tibial rotation using K-wires placed into the anterior margin of the tibia proximal and distal to the osteotomy. RESULTS: An overall mean of 4.4 ± 2.8° internal rotation of the distal tibia has been shown. In four patients with additional single step double bundle ACL-replacement after harvesting ipsilateral autologous hamstring grafts, the distal tibia rotated internally by 0.1 ± 0.3°, accordingly in the other 46 patients by 4.8 ± 2.6°. CONCLUSIONS: Valgus OW-HTO produces significant internal axial rotation of the distal tibia. This might be caused by soft tissue tension of the medial hamstrings/soft tissue structures and the location of the lateral tibial hinge. CLINICAL RELEVANCE: Surgeons have to take into consideration that valgus OW HTO might result in significant 3D changes of the tibia. Higher degrees of internal torsion of the tibia might influence overall gait mechanics and specifically alternate patellofemoral kinematics.

3.0T MR imaging of the ankle: Axial traction for morphological cartilage evaluation, quantitative T2 mapping and cartilage diffusion imaging-A preliminary study.

PURPOSE: To determine the impact of axial traction during high resolution 3.0T MR imaging of the ankle on morphological assessment of articular cartilage and quantitative cartilage imaging parameters. MATERIALS AND METHODS: MR images of n=25 asymptomatic ankles were acquired with and without axial traction (6kg). Coronal and sagittal T1-weighted (w) turbo spin echo (TSE) sequences with a driven equilibrium pulse and sagittal fat-saturated intermediate-w (IMfs) TSE sequences were acquired for morphological evaluation on a four-point scale (1=best, 4=worst). For quantitative assessment of cartilage degradation segmentation was performed on 2D multislice-multiecho (MSME) SE T2, steady-state free-precession (SSFP; n=8) T2 and SSFP diffusion-weighted imaging (DWI; n=8) images. Wilcoxon-tests and paired t-tests were used for statistical analysis. RESULTS: With axial traction, joint space width increased significantly and delineation of cartilage surfaces was rated superior (P<0.05). Cartilage surfaces were best visualized on coronal T1-w images (P<0.05). Differences for cartilage matrix evaluation were smaller. Subchondral bone evaluation, motion artifacts and image quality were not significantly different between the acquisition methods (P>0.05). T2 values were lower at the tibia than at the talus (P<0.001). Reproducibility was better for images with axial traction. CONCLUSION: Axial traction increased the joint space width, allowed for better visualization of cartilage surfaces and improved compartment discrimination and reproducibility of quantitative cartilage parameters.

Diverse expression of selected cytokines and proteinases in synovial fluid obtained from osteoarthritic and healthy human knee joints.

BACKGROUND: Osteoarthritis (OA) is defined by signs and symptoms of inflammation within the affected joint. The aim of this study is to determine the mRNA expression levels of selected cytokines and matrix-metalloproteinases of cells found in synovial fluid (SF) obtained from osteoarthritic knee joints compared to healthy controls. METHODS: SF was obtained from 40 patients undergoing total knee arthroplasty due to evident OA and from 10 healthy controls. Expression of TNF-α, IL-1ß, MMP-1 and MMP-3 was assayed among both groups by performing qPCR. Patients were configured concerning age, gender and BMI. RESULTS: IL-1ß, MMP-1 and MMP-3 showed significantly higher expression among the OA group compared to control (P < 0.001). Strong correlation appeared between expression of MMP-1 and MMP-3 among OA patients (r = 0.856); no correlation was found between age, gender or BMI and cytokine/proteinase expression. Expression of IL-1ß, MMP-1 and MMP-3 within SF was elevated in OA-patients. CONCLUSION: Consequently, cells within SF expressing cytokines and proteinases may play a relevant role in the progression of joint destruction. Considering the fact that SF in an OA joint comprises abnormal amounts of detrimental bioactive proteins, temporary clearance, dilution or suppression/modulation by means of lavage or disease-modifying medication may be promising to constitute interim relief or even postpone disease progression due to decreased inflammatory and/or degrading activity within the articular environment.

Early changes of trabecular bone structure in asymptomatic subjects with knee malalignment.

OBJECTIVE: To investigate whether alterations of trabecular bone structure can already be found in young asymptomatic subjects with knee malalignment. METHODS: Forty-eight subjects with neutral, mild varus, severe varus, and valgus knee joint alignment were included in this study (12 subjects in each group). Histomorphometric and texture parameters of the trabecular bone in the medial/lateral femur/tibia were determined using 1.5-T magnetic resonance imaging. RESULTS: Apparent trabecular thickness in the medial tibia compartment was lower in the valgus group (mean ± standard error, 0.353 ± 0.012 mm) compared with the neutral (0.396 ± 0.011 mm; P = 0.043), mild varus (0.403 ± 0.011 mm; P = 0.038) and severe varus groups (0.416 ± 0.013 mm; P = 0.015). In the medial femur compartment, fractal dimension was significantly greater in the mild (1.697 ± 0.005; P = 0.015) and severe varus groups (1.698 ± 0.005; P = 0.036) than in the valgus group (1.674 ± 0.005). CONCLUSIONS: The observed findings may be signs of the adaptation of the subchondral bone to altered loading conditions and possibly of early knee joint impairment.

Influence of extremely low frequency, low energy electromagnetic fields and combined mechanical stimulation on chondrocytes in 3-D constructs for cartilage tissue engineering.

Articular cartilage, once damaged, has very low regenerative potential. Various experimental approaches have been conducted to enhance chondrogenesis and cartilage maturation. Among those, non-invasive electromagnetic fields have shown their beneficial influence for cartilage regeneration and are widely used for the treatment of non-unions, fractures, avascular necrosis and osteoarthritis. One very well accepted way to promote cartilage maturation is physical stimulation through bioreactors. The aim of this study was the investigation of combined mechanical and electromagnetic stress affecting cartilage cells in vitro. Primary articular chondrocytes from bovine fetlock joints were seeded into three-dimensional (3-D) polyurethane scaffolds and distributed into seven stimulated experimental groups. They either underwent mechanical or electromagnetic stimulation (sinusoidal electromagnetic field of 1 mT, 2 mT, or 3 mT; 60 Hz) or both within a joint-specific bioreactor and a coil system. The scaffold-cell constructs were analyzed for glycosaminoglycan (GAG) and DNA content, histology, and gene expression of collagen-1, collagen-2, aggrecan, cartilage oligomeric matrix protein (COMP), Sox9, proteoglycan-4 (PRG-4), and matrix metalloproteinases (MMP-3 and -13). There were statistically significant differences in GAG/DNA content between the stimulated versus the control group with highest levels in the combined stimulation group. Gene expression was significantly higher for combined stimulation groups versus static control for collagen 2/collagen 1 ratio and lower for MMP-13. Amongst other genes, a more chondrogenic phenotype was noticed in expression patterns for the stimulated groups. To conclude, there is an effect of electromagnetic and mechanical stimulation on chondrocytes seeded in a 3-D scaffold, resulting in improved extracellular matrix production.

The dependence of autologous chondrocyte transplantation on varying cellular passage, yield and culture duration.

Matrix-assisted chondrocyte transplantation (m-ACI) still lacks any standardization in its execution in terms of cell passage (P), cell yield (C) and in vitro membrane-holding time (T). It was the goal of this study to analyze the effect of shifting cell culture parameters (P, C, T) on the in vitro as well as in vivo effort of a regulated animal m-ACI. Autologous rabbit knee articular chondrocytes were seeded within bilayer collagen I/III 3-D matrices in variation of P, C and T. Each time, 2 PCT-identical by 2 PCT-identical cell-matrix-constructs (CMC)/animal were created. Simultaneously 2 (PCT-distinct) were re-implanted (CMC-e) autologous into artificial trochlear pristine chondral defects in vivo to remain for 12 weeks while the remaining 2 were harvested (CMC-i) for immediate in vitro analysis at the time of transplantation of their identical twins. mRNA of both, CMC-e regenerates and CMC-i membranes, was analyzed for Collagen-1,-2,-10, COMP, Aggrecan, Sox9 expression by use of a mixed linear model, multiple regression analysis. Generally, CMC-i values were higher than CMC-e values for differentiation targets; the opposite was true for dedifferentiation targets. Regarding individual gene expression, in vivo regenerate cell-matrix properties were significantly dependent on initial cell-matrix in vitro values as a sign of linearity. The parameter membrane-holding time (T) had strongest effects on the resulting mRNA expression with slightly less impact of the parameter passage (P), whereas cell yield (C) had clearly less effects. Noting differences between in vitro and in vivo data, in general, optimal expression patterns concerning chondrogenic differentiation were achieved by few passages, medium cellular yield, short membrane-holding time. Clinical m-ACI may benefit from optimal orchestration of the cell culture parameters passage, yield and time.

Control of posterior tibial slope and patellar height in open-wedge valgus high tibial osteotomy.

BACKGROUND: Valgus-producing open-wedge high tibial osteotomy is an established treatment for varus malalignment and medial osteoarthritis, with reproducible results in the frontal plane. However, an undesirable but often accepted increase in posterior tibial slope and decrease in patellar height are still routinely seen. PURPOSE: To evaluate the influence of valgus open-wedge high tibial osteotomy on posterior tibial slope and patellar height when special techniques are used to minimize unwanted changes. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Twenty-five patients, 3 women and 22 men (mean age, 40.2 years), underwent valgus open-wedge high tibial osteotomy. Several technical steps were taken to prevent an increase in posterior tibial slope during the osteotomy. To minimize patellar height changes, the tibial tuberosity was left on either the proximal or distal fragment, depending on the desired patellofemoral effect. The medial and lateral posterior slope was measured using the proximal posterior cortex as a reference; the patellar height was assessed with the Caton-Deschamps Index and compared on preoperative and postoperative radiographs. RESULTS: No significant posterior tibial slope changes were observed. Patellar height increased with both types of tibial tuberosity osteotomy. With the proximal osteotomy, the Caton-Deschamps Index increased from 0.95 to 0.97; with the distal osteotomy, it increased from 0.89 to 0.95. The change was not significant with either osteotomy. The posterior tibial slope did not change on the medial side, measuring 4.2 preoperatively and postoperatively. The lateral slope decreased from 5.4 to 5.1. There was no correlation between the correction in the coronal plane and the changes in the sagittal plane. CONCLUSION: Open-wedge high tibial osteotomy can be performed without significant changes in patellar height or posterior tibial slope if specific intraoperative methods are used to prevent their occurrence. Analysis and control of sagittal changes in valgus open-wedge high tibial osteotomy should reduce the incidence of unwanted changes in patellar height and posterior tibial slope.

May smokers and overweight patients be treated with a medial open-wedge HTO? Risk factors for non-union.

PURPOSE: The purpose of this retrospective study was to investigate the rate of non-union after medial open-wedge high tibial osteotomy (HTO) with the Tomofix(®) plate. In addition, risk factors with a possible influence on the development of a non-union were analysed. METHODS: Between 2006 and 2008, a total of 186 medial open-wedge HTOs were performed in 182 patients. RESULTS: Ten cases of non-union (5.4%) were recorded. Risk factors with a statistically significant influence on the development of a non-union included smoking, body mass index and fracture of the lateral cortical hinge. No influence was detected for the factors age, degree of correction, concomitant diseases, postoperative complications, drug use, additionally performed procedures and use of a temporary lag screw. CONCLUSION: Within this study, it could be demonstrated that the rate of non-union in medial open-wedge HTO is not higher than in the lateral closed-wedge technique. Concerning the detected risk factors, the importance of the preservation of the lateral cortex is emphasised. In addition, it is necessary to discuss the risk of non-union with smokers and overweight patients when planning the therapy. Finally, it should be considered to perform iliac crest bone grafting in these high-risk patients a priori.

Ganglia of the tarsal sinus: MR imaging features and clinical findings.

PURPOSE: To analyze MR imaging and clinical findings associated with ganglia of the tarsal sinus. MATERIALS AND METHODS: In a record search, ganglia of the tarsal sinus were retrospectively identified in 26 patients (mean age 48±16 years), who underwent MR imaging for chronic ankle pain. Images were reviewed by two radiologists in consensus for size and location of ganglia, lesions of ligaments of the ankle and the tarsal sinus, tendon abnormalities, osteoarthritis, osseous erosions and bone marrow abnormalities. Medical records were reviewed for patient history and clinical findings. RESULTS: Ganglia were associated with the interosseus ligament in 81%, the cervical ligament in 31% and the retinacula in 46% of cases. Signal alterations suggesting degeneration were found in 85%, 50% and 63% in case of the interosseus ligament, the cervical ligament and the retinacula, respectively. Scarring of the anterior talofibular ligament and the fibulocalcaneal ligament was found in 68% and 72% of the patients, respectively, while only 27% of the patients recalled ankle sprains. Ganglia at the retinacula were highly associated with synovitis and tendinosis of the posterior tibial tendon (p<0.05). CONCLUSION: All patients with ganglia in the tarsal sinus presented with another pathology at the ankle, suggesting that degeneration of the tarsal sinus may be a secondary phenomenon, due to pathologic biomechanics at another site of the hind foot. Thus, in patients with degenerative changes of the tarsal sinus, one should be alerted and search for underlying pathology, which may be injury of the lateral collateral ligaments in up to 70%.