Carl Haasper

Influence of perfusion and cyclic compression on proliferation and differentiation of bone marrow stromal cells in 3-dimensional culture

Until now, there has been no in vitro model that duplicates the environment of bone marrow. The purpose of this study was to analyze proliferation and differentiation of human bone marrow stromal cells (hBMSC) under the influence of continuous perfusion and cyclic mechanical loading. hBMSC of seven individuals were harvested, grown in vitro, and combined. 10(6) hBMSC were seeded on a bovine spongiosa disc and incubated in a bioreactor system. Cell culture was continued using three different conditions: Continuous perfusion (group A), 10% cyclic compression at 0.5Hz (group B) and static controls (group C). After 24h, 1, 2, and 3 weeks, we determined cell proliferation (MTS-assay) and osteogenic differentiation (osteocalcin ELISA, Runx2 mRNA). Tenascin-C mRNA was quantified to exclude fibroblastic differentiation. In groups A and B, proliferation was enhanced after 2 weeks (48.6+/-19.6x10(3) (A) and 44.6+/-14.3 x 10(3) cells (B)) and after 3 weeks (46.6+/-15.1 x 10(3) (A) and 44.8+/-10.2 x 10(3) cells (B)) compared with controls (26.3+/-10.8 x 10(3) (2 weeks) and 17.1+/-6.5 x 10(3) cells (3 weeks), p<0.03). Runx2 mRNA was upregulated in both stimulated groups after 1, 2, and 3 weeks compared to control (group A, 1 week: 5.2+/-0.7-fold; p<0.01, 2 weeks: 4.4+/-1.9-fold; p<0.01, 3 weeks: 3.8+/-1.7-fold; p=0.013; group B, 1 week: 3.6+/-1.1-fold, p<0.01, 2 weeks: 4.2+/-2.2-fold, p<0.01; 3 weeks: 5.3+/-2.7-fold, p<0.01). hBMSC stimulated by cyclic compression expressed the highest amount of osteocalcin at all time points (1 week: 294.5+/-88.4 mg/g protein, 2 weeks: 294.4+/-73.3mg/g protein, 3 weeks: 293.1+/-83.6 mg/g protein, p0.03). The main stimulus for cell proliferation in a 3-dimensional culture of hBMSC is continuous perfusion whereas mechanical stimulation fosters osteogenic commitment of hBMSC. This study thereby contributes to the understanding of physical stimuli that influence hBMSC in a 3-dimensional cell culture system.

Influence of perfusion and compression on the proliferation and differentiation of bone mesenchymal stromal cells seeded on polyurethane scaffolds

In the present study, a porous meniscal-shaped scaffold consisting of polyurethane (PU)-based 1, 4-butanediisocyanate (BDI), which provided a 3-D culture condition for human bone mesenchymal stromal cells (hBMSC) was employed. A bioreactor was utilized to produce perfusion and mechanical stimulations. The viability, proliferation and fibro-cartilaginous differentiation of the hBMSC cultured on the PU-based meniscal scaffold were investigated during the perfusion and mechanical stimulation process. In addition, the mechanical properties of the cell-laden scaffolds were examined as well. Our finding indicated that the perfusion (10 ml/min) and on-off cyclic compressions mechanical stimulation (10% strain, 0.5 Hz, 4 times/day, 2 h/time with 4 h of rest thereafter) maintained the viability and promoted the proliferation of hBMSC over 2 weeks. The on-off cyclic compression caused a 1.85 fold increase in equilibrium modulus. Meanwhile, type I procollagen produced by hBMSC was increased for 3.02-fold after 2 weeks culture. On the other hand, the irrigating medium enhanced the synthesis of type III procollagen for 2.24-fold after 2 weeks. Tensile modulus was elevated for 2.02-fold in perfusion group after 1 week, which was decreased after 2 weeks unexpectedly. Our study suggests that the perfusion and on-off compression are promising to enhance the functional properties of the hBMSC-laden PU-based meniscal scaffold.

Collagen type I hydrogel allows migration, proliferation, and osteogenic differentiation of rat bone marrow stromal cells

Hydrogels are potentially useful for many purposes in regenerative medicine including drug and growth factor delivery, as single scaffold for bone repair or as a filler of pores of another biomaterial in which host mesenchymal progenitor cells can migrate in and differentiate into matrix-producing osteoblasts. Collagen type I is of special interest as it is a very important and abundant natural matrix component. The purpose of this study was to investigate whether rat bone marrow stromal cells (rBMSCs) are able to adhere to, to survive, to proliferate and to migrate in collagen type I hydrogels and whether they can adopt an osteoblastic fate. rBMSCs were obtained from rat femora and plated on collagen type I hydrogels. Before harvest by day 7, 14, and 21, hydrogels were fluorescently labeled, cryo-cut and analyzed by fluorescent-based and laser scanning confocal microscopy to determine cell proliferation, migration, and viability. Osteogenic differentiation was determined by alkaline phosphatase activity. Collagen type I hydrogels allowed the attachment of rBMSCs to the hydrogel, their proliferation, and migration towards the inner part of the gel. rBMSCs started to differentiate into osteoblasts as determined by an increase in alkaline phosphatase activity after two weeks in culture. This study therefore suggests that collagen type I hydrogels could be useful for musculoskeletal regenerative therapies.

Repair of a segmental long bone defect in human by implantation of a novel multiple disc graft

Large segmental defects of the weight bearing long bones are very difficult to reconstruct. Current treatment options are afflicted with several limitations and disadvantages. We describe a novel approach to regenerate a segmental long bone defect in a patient using a multiple disc graft. Decellularized bovine trabecular bone discs were seeded with autologous bone marrow cells and cultured in a perfusion chamber for three weeks. Multiple cell-seeded discs were implanted to close a 72 mm defect of the distal tibia in a 58-year-old woman, and fixed by an intramedullary nail. Bone formation was assessed non-invasively by plain radiographs and 18F-labeled sodium fluoride-based co-registration of positron emission- and computed tomography (PET/CT). Bone was actively formed around the grafted defect as early as six weeks after surgery. Because the tibia was sufficiently stabilized, the patient was able to freely walk with full weight bearing 6 weeks after surgery. The uneventful two-year follow-up and the satisfaction of the patient demonstrated the success of the procedure. Therefore the use of multiple cell-seeded disc grafts can be considered as a treatment alternative for patients with segmental long bone defects.

Biodegradable Screw Versus a Press-Fit Bone Plug Fixation for Hamstring Anterior Cruciate Ligament Reconstruction A Prospective Randomized Study

Background Press-fit fixation of a tendon graft has been advocated to achieve tendon-to-bone healing. Hypothesis Fixation of hamstring tendon grafts with a porous bone scaffold limits bone tunnel enlargement compared with a biodegradable interference screw fixation. Study Design Randomized controlled trial; Level of evidence, 1. Methods Between 2005 and 2006, 20 patients (17 men, 3 women) with a primary reconstruction of the anterior cruciate ligament (ACL) were enrolled in this study. Patients were randomized to obtain graft fixation in the tibial tunnel either by means of an interference screw (I) or a press-fit fixation with a porous bone cylinder (P). At 3 months after surgery, a computed tomography (CT) scan of the knee was performed, and tunnel enlargement was analyzed in the coronal and sagittal planes for the proximal, middle, and distal thirds of the tunnel. After 6 months and 1 and 2 years, radiographs of the knee in the sagittal and coronal plane were analyzed for bone tunnel widening. The International Knee Documentation Committee (IKDC), Tegner, and Lysholm scores of both groups were compared after 1 and 2 years. Results The bone tunnel enlargement determined by CT was 106.9% ± 10.9% for group P and 121.9% ± 9.0% for group I (P < .02) in the anteroposterior (AP) plane and 102.8% ± 15.2% versus 121.5% ± 10.1% in the coronal plane (P < .01). The IKDC, Tegner, and Lysholm scores improved in both groups from preoperatively to postoperatively without significant differences between the 2 groups. There was a trend to higher knee stability in group P after 3 months (0.6 ± 1.4 mm vs 1.8 ± 1.5 mm; P = .08). Conclusion Both interference screw and a press-fit fixation lead to a high number of good or very good outcomes after ACL reconstruction. Tibial press-fit fixation decreases the amount of proximal bone tunnel enlargement.

Alcohol intoxication in road traffic accidents leads to higher impact speed difference, higher ISS and MAIS, and higher preclinical mortality

Alcohol is one of the most important personal risk factors for serious and fatal injuries, contributing to approximately one third of all deaths from accidents. It is also described that alcohol intoxication leads to a higher mortality in the clinical course. In this study, we hypothesized that alcohol intoxication leads to different accident kinematics, a higher ISS (Injury Severity Score), and higher preclinical mortality compared to sober patients. A technical and medical investigation of alcohol intoxicated road users was performed on the scene of the crash and at the primary admitting hospital. Alcohol testing was performed with either breath alcohol tests or measurement of blood alcohol concentration (BAC) in a standard laboratory test. Between 1999 and 2010, 37,635 road traffic accidents were evaluated by the Accident Research Unit. Overall 20,741 patients were injured, 2.3% of the patients were killed. Among the injured patients, 2.2% with negative BAC were killed, compared to 4.6% fatal injuries in patients with a positive BAC (p < 0.0001). Of the patients with a positive BAC, 8.0% were severely injured, compared to 3.6% in the BAC negative group (p < 0.0001). Regarding the relative speed at impact (Δv for motorized drivers, vehicle collision speed for pedestrians and bikers), there was a significant higher difference for BAC positive patients (30 ± 20) compared to the BAC negative patients (25 ± 19, p < 0.0001). Alcohol intoxication in trauma patients leads to higher preclinical mortality, higher impact speed difference, and higher injury severity. The subgroup analysis for different alcohol concentrations shows no difference in ISS, MAIS, and relative speed, but a correlation of increasing age of patients with higher alcohol concentrations.

Prognostic value of procalcitonin (PCT) and/or interleukin-6 (IL-6) plasma levels after multiple trauma for the development of multi organ dysfunction syndrome (MODS) or sepsis

UNLABELLED Despite recent advances in treatment of severe injured patients, e.g. due to damage control orthopaedics, multi organ dysfunction syndrome (MODS) and sepsis are major complications in daily practice. During one year 94 patients were prospectively collected. INCLUSION CRITERIA ISS 16, age 18-60 y, primary admission to our level-1 trauma center, survival > 48 hours after trauma. The development of MODS and sepsis were observed and different groups were formed (+/-). Demographic data revealed no significant differences between the subgroups. Comparing groups +MODS and -MODS significant differences on admission day were observed, when PCT showed first on day 2 after trauma differences. Regarding the development of sepsis PCT was advantageous to IL-6 showing significant higher plasma levels in group +sepsis from the first day after trauma. Serum levels of IL-6 and PCT could be useful in early identification of high risk patients to develop posttraumatic MODS. For sepsis PCT is the better prognostic factor.

Postnatal maturation of tendon, cruciate ligament, meniscus and articular cartilage: a histological study in sheep.

Orthopaedic basic science data on immature skeletons are rare in the literature. Since the number of knee injuries in young humans is steadily increasing, studies on immature animals such as sheep, which can be used as model systems are becoming more and more important. However, no baseline data are available on physiologic and morphologic changes during growth in the relevant tissues. In the present study, histomorphometric changes in the tendon of the musculus flexor digitalis superficialis, the cranial cruciate ligament, the medial meniscus and the articular cartilage of the medial femoral condyle were identified in sheep between the ages of 1 and 40 weeks postnatally. Profound changes in tissue composition during growth could be observed. A high cellularity in the early postnatal period decreases to a constant lower level after 18 weeks. Similar changes during postnatal growth could be observed for blood vessel density. Also, staining of alpha-smooth muscle actin (SMA) and vascular endothelial growth factor (VEGF) steadily decreased. In contrast, the number of components of extracellular matrix steadily increased in all tissues. The age of 18 weeks seems to be a threshold after which the tissue composition of the observed structures remains constant in this species.

Histologic and biomechanical analysis of anterior cruciate ligament graft to bone healing in skeletally immature sheep.

PURPOSE It was our aim to establish an animal model and to investigate the tendon graft-to-bone and physis healing process in skeletally immature sheep after reconstruction of the anterior cruciate ligament (ACL). METHODS Thirty-two immature sheep aged 4 months underwent a fully transphyseal ACL reconstruction by use of a soft-tissue graft. The animals were subsequently killed after 3, 6, 12, and 24 weeks and analyzed histologically and biomechanically. RESULTS There was a transient hypertrophy of the physis tissue at the passing site of the graft. Anchoring Sharpey-like fibers evolved as early as 3 weeks after surgery. A strong expression of collagen III messenger ribonucleic acid within the first 6 weeks preceded this anchoring process. The maximum load to failure of the tendon graft in the reconstructed knees initially decreased to 37.8 +/- 17.8 N after 3 weeks and was restored to 522.9 +/- 113 N after 24 weeks. Tendon graft stiffness was restored to 86% when compared with the control knees. CONCLUSIONS The early anchoring by Sharpey fibers was found at 3 weeks with continued maturation to 24 weeks. This development of anchoring fibers corresponded to that of biomechanical strength, starting with 5% of the normal knee at 3 weeks and then 15.2% at 6 weeks, 41.2% at 12 weeks, and 69% at 24 weeks. Tendon graft-to-bone and physis healing in skeletally immature sheep is further characterized by a transient hypertrophy of the physis cartilage. The physis recovers well from the trauma of drilling and placement of a soft-tissue graft. The early development of Sharpey-like fibers results in a solid integration of the graft into bone in a timely manner. CLINICAL RELEVANCE ACL reconstruction in skeletally immature individuals is still controversial. This study describes in detail the histologic and biomechanical stages of tendon graft healing to the bone and physis. These data enrich the existing knowledge of previous studies in adult sheep and may provide a basis for further research in the controversial field of ACL reconstruction during growth.

The Abbreviated Injury Scale (AIS). Options and problems in application

The new AIS (Abbreviated Injury Scale) was released with an update by the AAAM (Association for the Advancement of Automotive Medicine) in 2008. It is a universal scoring system in the field of trauma applicable in clinic and research. In engineering it is used as a classification system for vehicle safety. The AIS can therefore be considered as an international, interdisciplinary and universal code of injury severity. This review focuses on a historical overview, potential applications and new coding options in the current version and also outlines the associated problems.ZusammenfassungDie AIS (Abbreviated Injury Scale) ist von der AAAM (Association for the Advancement of Automotive Medicine) 2008 mit einem Update herausgegeben worden. Sie ist eines der universellen Scoringwerkzeuge im unfallchirurgischen Fachgebiet mit Einsatzpotenzial in Klinik und Forschung. Die Ingenieurswissenschaften nutzen sie in der Fahrzeugtechnik zur Bewertung der Fahrzeugsicherheit. Damit kann die AIS als internationale, interdisziplinäre und universelle Klassifikation der Verletzungsschwere angesehen werden. Dieser Beitrag liefert einen Überblick zur Historie, Anwendungsmöglichkeit und verbesserten Kodiermöglichkeiten durch die neu erschienene Version. Dabei werden aber auch Probleme aufgezeigt.AbstractThe new AIS (Abbreviated Injury Scale) was released with an update by the AAAM (Association for the Advancement of Automotive Medicine) in 2008. It is a universal scoring system in the field of trauma applicable in clinic and research. In engineering it is used as a classification system for vehicle safety. The AIS can therefore be considered as an international, interdisciplinary and universal code of injury severity. This review focuses on a historical overview, potential applications and new coding options in the current version and also outlines the associated problems.