Eric Hesse

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.

Oncogenic osteomalacia: exact tumor localization by co-registration of positron emission and computed tomography.

In oncogenic osteomalacia, the causative tumor is almost always difficult to find. A novel diagnostic approach is presented that facilitates a precise and rapid localization of the associated lesion by PET‐CT co‐registration using the radiotracer 68Ga‐DOTANOC.

Double disruption of α2A- and α2C -adrenoceptors results in sympathetic hyperactivity and high-bone-mass phenotype

Evidence demonstrates that sympathetic nervous system (SNS) activation causes osteopenia via β2‐adrenoceptor (β2‐AR) signaling. Here we show that female mice with chronic sympathetic hyperactivity owing to double knockout of adrenoceptors that negatively regulate norepinephrine release, α2A‐AR and α2C‐AR (α2A/α2C‐ARKO), present an unexpected and generalized phenotype of high bone mass with decreased bone resorption and increased formation. In α2A/α2C‐ARKO versus wild‐type (WT) mice, micro–computed tomographic (µCT) analysis showed increased, better connected, and more plate‐shaped trabeculae in the femur and vertebra and increased cortical thickness in the vertebra, whereas biomechanical analysis showed increased tibial and femoral strength. Tibial mRNA expression of tartrate‐resistant acid phosphatase (TRACP) and receptor activator of NF‐κB (RANK), which are osteoclast‐related factors, was lower in knockout (KO) mice. Plasma leptin and brain mRNA levels of cocaine amphetamine–regulated transcript (CART), which are factors that centrally affect bone turnover, and serum levels of estradiol were similar between mice strains. Tibial β2‐AR mRNA expression also was similar in KO and WT littermates, whereas α2A‐, α2B‐ and α2C‐AR mRNAs were detected in the tibia of WT mice and in osteoblast‐like MC3T3‐E1 cells. By immunohistochemistry, we detected α2A‐, α2B‐, α2C‐ and β2‐ARs in osteoblasts, osteoclasts, and chondrocytes of 18.5‐day‐old mouse fetuses and 35‐day‐old mice. Finally, we showed that isolated osteoclasts in culture are responsive to the selective α2‐AR agonist clonidine and to the nonspecific α‐AR antagonist phentolamine. These findings suggest that β2‐AR is not the single adrenoceptor involved in bone turnover regulation and show that α2‐AR signaling also may mediate the SNS actions in the skeleton.

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

PURPOSEnIt 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).nnnMETHODSnThirty-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.nnnRESULTSnThere 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.nnnCONCLUSIONSnThe 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.nnnCLINICAL RELEVANCEnACL 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.

Femoral avulsion fracture of the posterior cruciate ligament in association with a rupture of the popliteal artery in a 9-year-old boy: a case report

Ruptures of the posterior cruciate ligament (PCL) and especially proximal bony avulsion fractures in children are very rare. This in combination with a rupture of the popliteal artery is extremely rare. Thus, an exact incidence is not available from the literature. Overall, these injuries are severe and often lead to chronic knee instability. We report a case of a 9-year-old boy who suffered a traumatic displacement of the left knee with a rupture of the popliteal artery. Prior to transfer to our department, he was treated by a saphenous vein bypass graft and by a transfixation of the knee using two oblique percutaneous pins. We performed magnetic resonance imaging (MRI) scan of the knee which revealed a femoral avulsion fracture of the PCL. Other ligaments and menisci were intact. A transosseous femoral fixation using non-absorbable stitches was carried out. A 1-year follow-up after surgery demonstrates intact peripheral perfusion and sensation, straight axes of both legs and a physiological gait. Minimal differences of the length and circumference of both legs could be measured. The posterior laxity (Lachman-test) was about 5/8xa0mm (right/left knee) and 2/5xa0mm (right/left knee) in 90° flexion. The range of motion (extension/flexion) was 5/0/140°–/5/100° (right–left knee). Intact cruciate ligaments were confirmed by MRI. Minimal experience exists in treatment of combined injuries to the PCL and the popliteal artery in children.

An animal model to study ACL reconstruction during growth

Controversy exists in the literature regarding the most appropriate treatment for anterior cruciate ligament (ACL) ruptures in the skeletally immature patient. To study the histological and biomechanical stages following ACL reconstruction during growth, it is necessary to establish an equivalent model to replicate the situation of an immature skeleton. In the present study we evaluated a large animal model of ACL reconstruction during growth using skeletally immature sheep. In contrast to existing models we did not intend to provoke growth disturbances. Instead, we identified those criteria known to be essential to prevent growth disturbances in the human situation and transformed them into an animal model. Applying those criteria to skeletally immature sheep at an age of four months, we performed a fully transphyseal ACL reconstruction of their right knee. The contralateral knee served as a control. The sheep were then sacrificed 3, 6, 12 and 24 weeks after surgery. This animal model is described in detail in the present manuscript. Basic science data are presented in order to 1) make it easier for other research groups to study ACL reconstruction during growth using our model and 2) to improve this model by modifying the one or other detail of the proposed technique. Using this model, remaining open questions concerning ACL reconstruction during growth can be addressed in future studies.

The Relation Between Tunnel Widening and Bone Mineral Density After Anterior Cruciate Ligament Reconstruction: An Experimental Study in Sheep

PURPOSEnThe aim of this study was to analyze the relation between bone mineral density (BMD) and femoral tunnel enlargement (TE) in a previously validated sheep model of soft-tissue anterior cruciate ligament (ACL) reconstruction.nnnMETHODSnThirty sheep underwent ACL reconstruction by use of a soft-tissue graft at the age of 4 months. Graft fixation was achieved with the EndoButton (Smith & Nephew Endoscopy, Andover, MA) and Suture Washer (Smith & Nephew Endoscopy). Six animals were killed at 0, 3, 6, 12, and 24 weeks postoperatively. Each ACL-reconstructed knee was examined both by computed tomography to analyze the bone tunnel cross-sectional area and by dual-energy x-ray absorptiometry to analyze BMD.nnnRESULTSnThere was a significant increase in tunnel cross-sectional area. BMD decreased significantly within the first 3 weeks after surgery and increased thereafter. A positive correlation between TE and BMD was found. However, a subgroup analysis showed that there is no influence of BMD on the development of a tunnel widening.nnnCONCLUSIONSnThe hypothesis that a TE would be associated with a loss in BMD was not confirmed. Tunnel widening during the first 6 months after ACL reconstruction is not affected by the transient changes in BMD.nnnCLINICAL RELEVANCEnThere is no correlation between TE and BMD in an experimental sheep model of ACL reconstruction. Translational investigations will determine whether this is also true in humans.

Oncofetal fibronectin mRNA is highly abundant in the blood of patients with papillary thyroid carcinoma and correlates with high-serum thyroid-stimulating hormone levels.

BACKGROUNDnPapillary thyroid carcinoma (PTC) cells express oncofetal fibronectin (onfFN) mRNA, which may be useful to detect circulating tumor cells. The objective of this study was to determine the fraction of PTC patients having onfFN mRNA in their peripheral blood and to determine if onfFN mRNA levels are correlated with the status of the disease or with thyroid-stimulating hormone (TSH) serum concentrations.nnnMETHODSnThis study included 95 PTC patients, who were previously treated by thyroidectomy and radioactive iodine administration. Patients were examined by cervical sonography, whole-body (131)I scintigraphy, thyroglobulin measurement, and onfFN mRNA quantification both when they were being treated with L-thyroxine (L-T4) and after L-T4 withdrawal. The mean value for onfFN mRNA in blood from 25 healthy subjects was used as control for the onfFN mRNA assay.nnnRESULTSnAfter L-T4 withdrawal, serum TSH levels rose and onfFN mRNA was found in the peripheral blood of 33 of 64 (52%) disease-free patients, 15 of 23 (65%) patients with local residual disease, and 6 of 8 (75%) patients with known local or distant metastases. Continuous administration of L-T4 repressed serum TSH. In this state none of 17 (0%) disease-free patients and 1 of 4 (25%) patients with local residual disease had an elevated onfFN mRNA level, and 2 of 4 (50%) patients with metastasis had positive tests for serum onfFN mRNA.nnnCONCLUSIONSnonfFN transcripts are highly abundant in the peripheral blood of patients with PTC. L-T4 withdrawal, which produced elevated serum TSH concentrations in these athyroidic patients, markedly increased the fraction with positive tests for serum onfFN mRNA at all stages of the disease. Analyzing onfFN mRNA in the absence of a TSH stimulus allows a much better discrimination of different states of PTC disease and, based on current concepts of the significance of circulating mRNA, may be a useful tool to detect circulating thyroid cancer cells.

Injury situation of novice drivers in road traffic - a medical and technical analysis

AIMnNovice drivers are at high risk for crash involvement. We performed an analysis of causations, injury patterns and distributions of novice drivers in cars and on motorcycles in road traffic as a basis for proper measurements.nnnMETHODnData of accident and hospital records of novice drivers (licence < 2 years) were analysed focusing on the following parameters: injury type, localisation and mechanism, abbreviated injury scale (AIS), maximum AIS (MAIS), delta-v, collision speed and other technical parameters and compared with those of experienced drivers.nnnRESULTSnIn 18 352 accidents in the area of Hannover (years 1985-2004), 2602 novice drivers and 18 214 experienced drivers were recorded as having an accident. Novice car drivers were more often and more severely injured whereas on motorcycles the experienced drivers were at higher risk. Novice drivers of both groups sustained more often extremity injuries. 4.5 % of the novice car drivers were not restrained by seatbelts as compared to 3.7 % of the experienced drivers and 6.1 % of the novice motorcycle drivers did not wear a proper helmet (versus 6.5 %). Severe injuries were sustained in 20 % at collision speeds below 30 km/h and in 80 % at collision speeds above 50 km/h. Novice car drivers drove significantly older cars. The risk profile of novice drivers is similar to that of drivers older than 65.nnnCONCLUSIONnStructural protection and special lectures like skidding courses could be proper measurements next to harder punishment of violations.