Carsten Englert

Bonding of articular cartilage using a combination of biochemical degradation and surface cross-linking

After trauma, articular cartilage often does not heal due to incomplete bonding of the fractured surfaces. In this study we investigated the ability of chemical cross-linkers to facilitate bonding of articular cartilage, either alone or in combination with a pre-treatment with surface-degrading agents. Articular cartilage blocks were harvested from the femoropatellar groove of bovine calves. Two cartilage blocks, either after pre-treatment or without, were assembled in a custom-designed chamber in partial apposition and subjected to cross-linking treatment. Subsequently, bonding of cartilage was measured as adhesive strength, that is, the maximum force at rupture of bonded cartilage blocks divided by the overlap area. In a first approach, bonding was investigated after treatment with cross-linking reagents only, employing glutaraldehyde, 1-ethyl-3-diaminopropyl-carbodiimide (EDC)/N-hydroxysuccinimide (NHS), genipin, or transglutaminase. Experiments were conducted with or without compression of the opposing surfaces. Compression during cross-linking strongly enhanced bonding, especially when applying EDC/NHS and glutaraldehyde. Therefore, all further experiments were performed under compressive conditions. Combinations of each of the four cross-linking agents with the degrading pre-treatments, pepsin, trypsin, and guanidine, led to distinct improvements in bonding compared to the use of cross-linkers alone. The highest values of adhesive strength were achieved employing combinations of pepsin or guanidine with EDC/NHS, and guanidine with glutaraldehyde. The release of extracellular matrix components, that is, glycosaminoglycans and total collagen, from cartilage blocks after pre-treatment was measured, but could not be directly correlated to the determined adhesive strength. Cytotoxicity was determined for all substances employed, that is, surface degrading agents and cross-linkers, using the resazurin assay. Taking the favourable cell vitality after treatment with pepsin and EDC/NHS and the cytotoxic effects of guanidine and glutaraldehyde into account, the combination of pepsin and EDC/NHS appeared to be the most advantageous treatment in this study. In conclusion, bonding of articular cartilage blocks was achieved by chemical fixation of their surface components using cross-linking reagents. Application of compressive forces and prior modulation of surface structures enhanced cartilage bonding significantly. Enzymatic treatment in combination with cross-linkers may represent a promising addition to current techniques for articular cartilage repair.

Characterization of esterified hyaluronan-gelatin polymer composites suitable for chondrogenic differentiation of mesenchymal stem cells

Composite scaffolds of homogeneously mixed esterified hyaluronan (HY) and gelatin (G) were manufactured with variable component compositions (HY100%; HY95%/G5%; HY70%/G30%). The goals of this study were to analyze the produced composite scaffolds using physical and chemical methods, for example, scanning electron microscopy, IR-spectroscopy, water contact angle, protein assay, and tensile testing as well as to assess the effects of adding gelatin to the composite scaffolds on attachment, proliferation, and chondrogenic differentiation of human mesenchymal stem cells. Numbers of attached cells were significantly higher on the composite material compared to pure hyaluronan at different time points of two-dimensional or three-dimensional cell culture (p< 0.02). In composite scaffolds, a significantly greater amount of cartilage-specific extracellular matrix components was deposited after 28 days in culture (glycosaminoglycan: p < 0.001; collagen: p < 0.001) as compared with 100% hyaluronan scaffolds. Additionally, gelatin-containing composite scaffolds displayed stronger promotion of collagen type II expression than pure hyaluronan scaffolds. The mechanism, based on which gelatin influences cell adhesion, was examined. The effect was inhibited by collagenase treatment of the composites or by addition of alpha5beta1-integrin blocking antibodies to the cell suspension. In summary, the results describe the establishment of a class of composite polymer scaffolds, consisting of esterified hyaluronan and gelatin, which are potentially useful for cell-based tissue engineering approaches using mesenchymal stem cells for chondrogenic differentiation.

Synergistic effects of growth and differentiation factor-5 (GDF-5) and insulin on expanded chondrocytes in a 3-D environment

OBJECTIVE To investigate the effects of growth and differentiation factor-5 (GDF-5) alone or in combination with insulin on engineered cartilage from primary or expanded chondrocytes during 3-dimensional in vitro culture. DESIGN Juvenile bovine chondrocytes were seeded either as primary or as expanded (passage 2) cells onto polyglycolic acid fiber meshes and cultured for 3 weeks in vitro. Additionally, adult human chondrocytes were grown in pellet culture after expansion (passage 2). The culture medium was supplemented either with GDF-5 in varying concentrations or insulin alone, or with combinations thereof. RESULTS For primary chondrocytes, the combination of GDF-5 and insulin led to increased proliferation and construct weight, as compared to either factor alone, however, the production of glycosaminoglycans (GAG) and collagen per cell were not affected. With expanded bovine chondrocytes, the use of GDF-5 or insulin alone led to only very small constructs with no type II collagen detectable. However, the combination of GDF-5 (0.01 or 0.1 microg/ml) and insulin (2.5 microg/ml) yielded cartilaginous constructs and, in contrast to the primary cells, the observed redifferentiating effects were elicited on the cellular level independent of proliferation (increased production of GAG and collagen per cell, clear shift in collagen subtype expression with type II collagen observed throughout the construct). The synergistic redifferentiating effects of the GDF-5/insulin combination were confirmed with expanded adult human cells, also exhibiting a clear shift in collagen subtype expression on the mRNA and protein level. CONCLUSIONS In combination with insulin, GDF-5 appears to enable the redifferentiation of expanded chondrocytes and the concurrent generation of cartilaginous constructs. The demonstration of these synergistic effects also for adult human chondrocytes supports the clinical relevance of the findings.

Thoracic spondylitis from a mycotic (Streptococcus pneumoniae) aortic aneurysm: a case report.

Study Design. We report on a 54-year-old man with chronic lower back pain after recent streptococcus pneumoniae pulmonary infection, resulting in a mycotic aortic aneurysm and spondylodiscitis of the eighth vertebrae 6 months later. Successful surgical treatment and recurrence-free survival after 4 years are described. Summary of Background Data. Osteomyelitis by Streptococcus pneumoniae of the spine combined with contained rupture of a mycotic aortic aneurysm into lung and spine has not been reported to date. Mycotic aneurysms with pulmonary fistulas are reported to carry a mortality rate of up to 100%. Few cases have been reported with different operative and conservative strategies. Methods. The mycotic aortic aneurysm was excised using extracorporeal circulation and replaced by a Dacron graft. The spondylitic section of the eighth thoracic vertebrae was radically resected, and a tricortical bone block from the iliac crest was inserted into the defect. To keep compartments separated, collagen sponges with antibiotic supplementation were used. A triple antibiotic therapy (Metronidazol 3 × 0.5 g/day, Cefotaxim 3 × 2 g/day, and Flucloxacillin 3 × 2 g/day) was prescribed for 6 weeks and changed to Clindamycin for 1 year thereafter. Results. The patient made a good recovery and is free of recurrence 4 years after surgery. Conclusions. Lower back pain might be a projected pain. Particularly in older patients or in the presence of comorbidities resulting in an immunocompromised status, an aggressive workup may be indicated. Radical resection of inflammatory tissues, sparse use of implant material, and prolonged administration of antibiotics proved a successful strategy in this patient.

Elbow Dislocations: A Review Ranging from Soft Tissue Injuries to Complex Elbow Fracture Dislocations

This review on elbow dislocations describes ligament and bone injuries as well as the typical injury mechanisms and the main classifications of elbow dislocations. Current treatment concepts of simple, that is, stable, or complex unstable elbow dislocations are outlined by means of case reports. Special emphasis is put on injuries to the medial ulnar collateral ligament (MUCL) and on posttraumatic elbow stiffness.

Post-traumatic fulminant paradoxical fat embolism syndrome in conjunction with asymptomatic atrial septal defect: a case report and review of the literature

IntroductionFat embolism syndrome with respiratory failure after intramedullary nailing of a femur fracture is a rare but serious complication in trauma patients.Case presentationWe present the case of a 20-year-old Caucasian man who experienced paradoxical cerebral fat embolism syndrome with fulminant progression after intramedullary nailing of a femur fracture, in conjunction with a clinically asymptomatic atrial septal defect in a high position resulting in a right-to-left shunt.ConclusionFat embolism syndrome may occur as a fulminant complication following femoral fracture repair in the presence of a concomitant atrial septal defect with right-to-left shunt. Thus, in patients with cardiac right-to-left shunts, femurs should not be nailed intramedullary, not even in cases of isolated injuries.

Hypoxic downregulation of cellular proliferation and loss of phenotype stability in human osteoblasts is mediated by HIF-1α

Both, skeletal development and fracture healing depend on an orchestrated sequence of cellular growth and differentiation processes. Regional changes in tissue oxygen tension were proposed as key regulators of osteoblast proliferation and phenotype. Hypoxia results in the stabilization of hypoxia-inducible factor-1α (HIF-1α), thus influencing expression of a multitude of genes required for cellular adaptation. In the present study we dissected the effects of HIF-1α on cellular proliferation and gene expression of primary human osteoblasts. Primary human osteoblasts were studied by transfecting siRNA and plasmids coding for human HIF-1α. Gene expression was analyzed by western blot and quantitative PCR. Functional assays were performed to study HIF-1α function, i.e. proliferation and cell cycle analysis. As previously reported exposure to hypoxia led to a stabilization of HIF-1α on protein level and resulted in reduced rates of proliferation and osteocalcin expression. Furthermore, the expression of the proproliferative gene survivin was significantly reduced (p < 0.01). Knock down of HIF-1α attenuated hypoxic downregulation of proliferation (p < 0.05), and osteocalcin (p < 0.05) as well as survivin (p < 0.05) expression significantly. Importantly, the isolated overexpression of HIF-1α impaired proliferative activity and led to significantly reduced rates of expression of osteocalcin (p < 0.05) and survivin (p < 0.01). The present study shows that HIF-1α might reduce proliferation and survivin expression in primary human osteoblasts independently from cellular hypoxia. Furthermore, HIF-1α promoted the loss of the characteristic osteoblastic marker, osteocalcin in vitro. These findings underline the important role of HIF-1α in bone physiology and pathophysiology. Modulating HIF-1α function in hypoxic environments could be of value for future therapeutic approaches.

Influence of rotator cuff tears on glenohumeral stability during abduction tasks.

One of the main goals in reconstructing rotator cuff tears is the restoration of glenohumeral joint stability, which is subsequently of utmost importance in order to prevent degenerative damage such as superior labral anterior posterior (SLAP) lesion, arthrosis, and malfunction. The goal of the current study was to facilitate musculoskeletal models in order to estimate glenohumeral instability introduced by muscle weakness due to cuff lesions. Inverse dynamics simulations were used to compute joint reaction forces for several static abduction tasks with different muscle weakness. Results were compared with the existing literature in order to ensure the model validity. Further arm positions taken from activities of daily living, requiring the rotator cuff muscles were modeled and their contribution to joint kinetics computed. Weakness of the superior rotator cuff muscles (supraspinatus; infraspinatus) leads to a deviation of the joint reaction force to the cranial dorsal rim of the glenoid. Massive rotator cuff defects showed higher potential for glenohumeral instability in contrast to single muscle ruptures. The teres minor muscle seems to substitute lost joint torque during several simulated muscle tears to maintain joint stability. Joint instability increases with cuff tear size. Weakness of the upper part of the rotator cuff leads to a joint reaction force closer to the upper glenoid rim. This indicates the comorbidity of cuff tears with SLAP lesions. The teres minor is crucial for maintaining joint stability in case of massive cuff defects and should be uprated in clinical decision‐making.

Arthroscopic three dimensional autologous chondrocyte transplantation with navigation-guided cartilage defect size assessment

IntroductionThe treatment of large full thickness cartilage defects with matrix guided autologous chondrocyte transplantation shows promising results. However, in many cases an arthrotomy is needed to implant the cell seeded scaffolds. Recently techniques have been developed for arthroscopically guided ACT implantation. Correct defect mapping, to assess size and depth of the chondral lesions, and precise scaffold preparation and fixation are crucial for successful chondrocyte transplantation and remain to be not sufficiently optimized.MethodIn the present study, the geometries of two cartilage defects in cadaver knees were three times assessed, measured and transferred to biodegradable scaffolds with a navigation system by three different executors. The scaffolds were arthroscopically implanted into the cartilage defects.ResultsThe cartilage defect assessment was reproducible between all executors for all defect geometries. The implanted scaffolds showed a correct defect filling.ConclusionThe study showed the feasibility of an arthroscopic implantation of scaffolds for autologous chondrocytes transplantation. Navigation was a useful tool to exactly assess the cartilage defect geometry and allowed a precise transfer of navigated cartilage defect geometries for individualized scaffold preparation. Navigation can help to accomplish and optimize arthroscopically guided chondrocyte transplantations.

Amplifikationsoptimierung humaner Chondrozyten durch Wachstumsfaktoren

ZusammenfassungGrundlagen: Knorpelläsionen stellen in der rekonstruktiven Chirurgie ein noch ungelöstes Problem dar. Eine vielversprechende Therapiemöglichkeit könnte die in-vitro-Herstellung hyalinen Knorpels aus autolog entnommenen Chondrozyten mit anschließender Implantation in den Defekt darstellen. Methodik: Humane Chondrozyten wurden unter folgenden Bedingungen kultiviert: Gruppe 1 (Kontrollgruppe): Kultivierung in 25 cm2 Zellkulturflaschen (Costar) mit RPMI-Medium (6 %-AB-Serum, L-Glutamin, Hepes-Puffer und Antibiotikazusatz); Gruppe 2: Zugabe von unterschiedlichen Konzentrationen an IGF-I (1 ng/ml, 10 ng/ml) zu RPMI-Medium; Gruppe 3: Kultivierung wie bei Kontrollgruppe mit additiver Beschichtung der Zellkulturflaschen mit unterschiedlichen Konzentrationen an RGD (5 µg/ml; 7,5 µg/ml; 10 µg/ml; 20 µg/ml); Gruppe 4: Kombination von RGD-Beschichtung (5 µg/ml, 10 µg/ml) und Zugabe von IGF-I (1 ng/ml; 10 ng/ml) zum Medium. Ergebnisse: In der Kontrollgruppe war innerhalb von 2 Wochen eine Verdopplung der Chondrozytenzellzahl möglich. In den Versuchsgruppen 2 und 3 konnte eine Verbesserung der Amplifikationsrate mit folgenden Maxima erzielt werden: Gruppe 2 (5 µg/ml RGD) 3, 1 fache und Gruppe 3 (1 ng/ml IGF-I) 2,6-fache Ausgangszellzahl. In Gruppe 4 (RGD und IGF-I) zeigten sich additive Effekte bei einer 4, 1 fachen Zellzahlvermehrung nach 14 Tagen. Durch RGD und IGF-I (Gruppen 2–4) wurde eine schnellere Dedifferenzierung und Adhäsion der Zellen am Kulturflaschenboden erreicht. Schlußfolgerung: Durch den kombinierten Einsatz von Wachstumsfaktoren (RGD/IGF-I) läßt sich die Zellzahl im Vergleich zur Nullprobe — im gleichen Zeitraum — mehr als verdoppeln, so daß die zur Züchtung hyalinen Knorpels notwendige Patientenprobe auf die Hälfte reduziert werden kann.SummaryBackground: Defects of cartilage are — by now — an unsolved problem. By using tissue-engineering, hyaline cartilage could be produced out of autologous chondrocytes in vitro and could be re-implanted into the defects. Methods: Human chondrocytes were incubated under following conditions: Group 1 (control group): Incubation in 25 cm2 cell culture flasks (Costar) with RPMI-medium (6%-AB-serum, L-Glutamin, Hepes-buffer and antibiotics); Group 2: Different concentrations of IGF-I (1 ng/ml, 10 ng/ml) were added to the RPMI-medium; Group 3: Incubation (like control group) with additional coating of the cell culture flasks with different concentrations of RGD (5 µg/ml; 7,5 µg/ml; 10 µg/ml; 20 µg/ml); Group 4: Combination of coating with RGD (5 µg/ml; 10 µg/ml) and addition of IGF-I (1 ng/ml; 10 ng/ml) to the medium. Results: The cells of the control group could be doubled within 2 weeks. The amplification rate of group 2 and 3 was improved in comparison to group 1 with the following maxima: Group 2 (5 µg/ml RGD) 3, 1 times and group 3 (1 ng/ml IGF-I) 2,6 times of the number of the cells in the beginning. Group 4 (RGD and IGF-I) showed additive effects with an increase in cell number of 4.1 times in comparison to the beginning. RGD and IGF-I (group 2 to 4) made an earlier dedifferentiation and adhesion of the cells to the bottom of the cell culture flasks possible. Conclusions: By using both growth factors (RGD and IGF-I), the number of cells could be enhanced more than 2 times in comparison to the control group within the same time. So less than half of the autologous patient’s cartilage is necessary for cultivation of hyaline cartilage.