Cholinergic nerve fibers in bone defects of a rat osteoporosis model and their regulation by implantation of bone substitution materials.

OBJECTIVES: Bone is innervated by autonomic nervous system that consists of sympathetic and parasympathetic nerves that were recently identified in bone. Thus we asked whether parasympathetic nerves occur in bone defects and at the interface of substitution materials that were implanted for stabilization and improvement of healing in an osteoporosis animal model. METHODS: Osteoporosis was induced in rats by ovariectomy and deficiency diet. A wedge-shaped osteotomy was performed in the metaphyseal area of femur. Eight different implants were inserted that were based on calcium phosphate cement, iron, silica-mineralized collagen, and modifications with strontium. Nerves were identified by immunohistochemistry with antibodies against vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH) and protein gene product 9.5 (PGP 9.5) as neuronal marker. RESULTS: Cholinergic nerves identified with VAChT immunostaining were detected in defects filled with granulation tissue and in surrounding mast cells. No immunolabeling of cholinergic nerves was found after implantation. The general presence of nerves was reduced after implantation as shown by PGP 9.5. Sympathetic nerves identified by TH immunolabeling were increased in strontium functionalized materials. CONCLUSION: Since cholinergic innervation was diminished after implantation a further increase in the compatibility of substitution materials to nerves could improve defect healing especially in osteoporotic bone.

[The simultaneous distally based sural flap. A therapeutic option for coverage of both heels]. / Der simultane distal gestielte Suralislappen. Therapieoption zur Defektdeckung beider Fersen.

Exposed bradytrophic tissue in regions with high mechanical loading is an indication for defect coverage with (myo-, adipo-) fasciocutaneous flaps. In this case, distally based sural flaps were used for bilateral coverage of defects in weight-bearing areas of feet after fourth-degree frostbite. Residual defects can be covered with a split skin mesh graft. The definitive prosthetic supply of the foot assumes a stabilized plantar soft tissue situation. Among its advantages in comparison to free microvascular flaps, the locally based fasciocutaneous flap can be harvested with less donor site morbidity after elevation and does not require secondary debulking. It has been shown that the reduced stability at the border zone between flap and mesh graft has an adverse effect.

[Distal radius fracture - a goalkeepers' injury in children and adolescents]. / Die Distale Radiusfraktur - eine Torwartverletzung bei Kindern und Jugendlichen.

INTRODUCTION: Fractures of the distal radius are the most frequent fractures of longbones during childhood and adolescence. A rare mechanism of injury is the attempt of a soccer goalkeeper to save the ball, shot with high velocity. MATERIAL AND METHODS: Reports of all children and adolescents up to the age of 14, suffering from a distal radius fracture from a three year period were evaluated retrospectively and this particular mechanism of injury was identified. All patients were reinvestigated after at least 10 months clinically. RESULTS: Amongst 516 distal radius- and distal forearm fractures, there were 213 (41.3 %) sports injuries. 61(28.6 %) of them resulted from playing soccer, with 48 (78.7 %) after a fall. 13 distal radius fractures were identified as a goalkeepers injury by a sharply shot ball. We found 6 buckle fractures, 4 complete fractures, 2 physeal separations Salter II and 1 metaphyseal greenstick fracture. At the time of the last follow up investigation all injured had regained their level of sports. DISCUSSION: Injuries to the lower extremities are most frequent in soccer. The ball itself may lead to fractures and dislocations of the hand and finger skeleton, even not unfrequently to distal radius fractures. Wrist protectors and an adequate behaviour in game and training may contribute to their avoidance.

Expression of EDA+ and EDB+ fibronectin splice variants in bone.

The extracellular matrix component fibronectin (fn) has fundamental functions in cell attachment, differentiation, proliferation, and migration. Isoforms of cellular fibronectin, named EDA+ fibronectin or embryonal EDB+ fibronectin, are generated by alternative splicing of its mRNA precursors. Little is known about the expression of EDA+ and EDB+ fibronectin splice variants in human bone. The aim of this study was to investigate the expression pattern of fibronectin splice variants in bone cell lines and in different human bone tissue samples (mature bone, early stages of fracture healing, hypotrophic nonunion, osteosarcoma). Analysis was done by immunostaining with recombinant and monoclonal antibodies, qualitative RT-PCR and LightCycler-based real-time quantitative RT-PCR assay. In osteoblast and osteosarcoma cell lines, abundant expression of EDA+ and EDB+ fibronectin was found in immunocytochemistry. High transcription levels of both splice variants mRNA were seen in quantitative RT-PCR in osteosarcoma cell lines. In mature bone, EDA+ and EDB+ were not detectable in immunohistochemistry. Transcription of mRNA in both splice variants was absent in these samples. Early stages of fracture healing and osteosarcoma cell samples exhibited extensive staining for EDA+ and EDB+ fibronectin, and high mRNA levels were found. Both osteosarcoma and bone fracture healing tissue expressed high mRNA levels of the fibronectin splice variants independent of benign or malignant behavior. Low level of EDA+ fibronectin mRNA transcription and focal immunohistochemical staining of EDA+ fibronectin was found in hypotrophic nonunions, whereas EDB+ fibronectin was not detected by immunohistochemistry and qualitative or quantitative PCR. EDA+ fibronectin was found in granulation tissue-forming processes in bone independent from bone-forming activity. EDB+ fibronectin was seen only in high-turnover new osteoid-forming processes like early stages of fracture healing and osteosarcoma and was absent in low-turnover processes like mature bone and hypotrophic nonunion. Both EDA+ and EDB+ fibronectin mark active processes in bone without differentiation between malignant or benign activity. In conclusion, EDA+ and EDB+ fibronectin splice variants are strong markers for active fibrogenetic and osteoid-forming processes in human bones.

In vivo mechanisms of hydroxyapatite ceramic degradation by osteoclasts: fine structural microscopy.

In the present study the in vivo mechanism of calcium-phosphate (CaP) ceramic degradation has been investigated by means of transmission electron microscopy. The results revealed osteoclast-mediated degradation of hydroxyapatite ceramic implanted into sheep bone by simultaneous resorption and phagocytosis. After 6 weeks of implantation, osteoclasts were localized immediately beneath the ceramic surface. They had formed resorption lacunae and exhibited typical ultrastructural features, such as the ruffled border, the clear zone, and the dorsal microvilli. Their resorption capacity also had become evident by alterations of the electron density and the shape of the CaP crystals localized within the acidic microenvironment of the ruffled border. Moreover, the osteoclasts simultaneously were capable of phagocytosing the resorbed CaP crystals. The formation of endophagosomes was performed (1) by the uptake of particles into large intracellular vacuoles, which were generated by deep invagination of the membranes of the osteoclastic ruffled border, and (2) by the encircling of particles due to the development of pseudopodia-like plasmaprotrusions of the ruffled border. The formation of endophagosomes was followed by the in situ fragmentation of the inclusion material, which subsequently was released into the extracellular space and phagocytosed by macrophages.

Effects of platelet growth factors on human mesenchymal stem cells and human endothelial cells in vitro.

The aim of the present in vitro study has been to investigate the effects of a enriched platelet derived growth factors on proliferation and migration of human endothelial and mesenchymal stem cells and on osteogenic differentiation of stem cells. Platelet rich plasma has been produced, yielding a four time higher number of thrombocytes than normal plasma. Degranulation of platelets has been performed by means of calcium and thrombin. Plasma has served as a control, whereas plasma in combination with calcium and thrombin was used to distinguish the difference between calcium and/or thrombin mediated effects and growth factor induced effects on the cells. The observed enhanced proliferation and migration of endothelial cells towards the platelet derived growth factors was driven by the plasma component of these preparations. However PDGF solely stimulated the migration and proliferation of mesenchymal stem cells. The increased osteogenic differentiation of growth factor treated mesenchymal stem cells was mostly driven by the high level of calcium used for the platelets degranulation. In summery, the different components of platelet derived growth factors work together to influence human endothelial and mesenchymal stem cells. This is of special clinically interest regarding the stimulation of bone healing in orthopaedic and traumatic surgery.

[Long-term results in the surgical treatment of pilon tibial fractures. A retrospective study]. / Langzeitergebnisse nach operativer Versorgung von Pilon-tibial-Frakturen. Eine retrospektive Untersuchung.

BACKGROUND: While Pilon fractures of the tibia have been treated for decades by primary open reduction and internal fixation by plate osteosynthesis, during the last 10 years differential treatment was developed: After primary open reduction nowadays patients are treated with (according to type of fracture and tissue damage). As well as primary open reduction and internal fixation a two-step treatment (primary external fixator and delayed ORIF) or consolidation by external fixator combined with minimal invasive osteosynthesis (cannulated screws and K-wires) has been implemented. Furthermore, the significance of primary bone grafting in comminuted fractures to prevent aseptic pseudarthrosis has been acknowledged. METHODS: Of 151 patients with 160 pilon fractures treated from January 1979 to May 1995, 107 patients (113 fractures) were evaluated. Only the results of C2 and C3 fractures could be compared, as only in these groups were all three types of treatment used. RESULTS: Over 75% of the treated fractures were closed fractures, most of them being fractures with a soft tissue damage grade 2 of the Oestern and Tscherne classification. In the open fractures we found mainly grade 3 fractures according to the Gustilo and Anderson classification. In 54.9% of all pilon tibial fractures we observed an uncomplicated course of healing. Early complications (25.7%) were mainly soft tissue infections, whereas we found pseudarthrosis to be the most frequent late complication. Highest infection rate (55.5%) was in the two-step treatment group (primary external fixator and delayed ORIF) and lowest in the primary internal stabilization group, although especially in the C2 and C3 fractures best clinical late results were obtained with the two-step procedure. CONCLUSION: The complication rate in the treatment of pilon fractures depends mainly on the type of fracture, the soft tissue damage and the type of treatment. The results of primary ORIF varied. In the case of low-grade soft tissue damage, good to excellent results were accomplished. In the case of higher-grade soft tissue damage, the problem of soft tissue coverage and reconstruction of the joint surface could be solved with good results by the two-step treatment. Herewith it is important to use limited open reduction of displaced fragments and fixation by cannulated screws and K-wires. We consider ORIF of the fibula necessary as stabilization of the second column of the ankle joint.

[The frozen shoulder. Arthroscopy, histological findings and transmission electron microscopy imaging]. / Arthroskopische, histologische und elektronenmikroskopische Untersuchungen.

INTRODUCTION: During a period of 17 months (February 1998 to July 1999) arthroscopy was performed in 172 patients because of pathologic changes of the shoulderjoint. METHODS: From the history and clinical examination, only eleven patients fulfilled the criteria for primary frozen shoulder set up by Lundberg in 1969. Further investigations such as X-ray examination, ultrasonography and MRI scanning showed no changes that could explain the clinical picture of shoulder stiffness. Of the eleven patients with frozen shoulder. additionally in two cases hyperthyroidism, in five cases diabetes type II and in a further five cases a Dupuytren's contracture could be found. Based on the operative procedure, recently published by Habermeyer, Ogilvie and Warner we carried out an arthroscopic arthrolysis of the stiff shoulder. RESULTS: Diagnostic arthroscopy demonstrated synovialitis in the upper and ventral areas of the joint in over 50% of the patients. Interestingly, intraarticular adhesions were not found in any of our patients; in particular, no obliteration of the axillary recess could be seen. Altogether, in seven of the eleven patients with frozen shoulder, a notably contracted biceps tendon was found which obviously applied increased the force on the humeral head, as in corresponding areas cartilage damage could be demonstrated arthroscopically. A significant increase in fibroblast cells next to loose areas of connective tissue could be shown in histological examinations of biopsies of the capsule in the rotatorer interval. We noted thickened vessel walls and an augmentation of the synovial surface, whereas typical inflammation could not be demonstrated in any of the histological specimens. In the transmission-electron microscope examination (TEM) of samples taken from the capsule in the rotatorer interval, structural changes in collagenous tissue were discernible. Typically, a loss of fibril order and a twisting of collagen fibrils were seen. This twirling led to an up to fourfold diameter of the collagenous fibrils, whereby thin, elastin-like filaments could be seen between the thickened bundles of collagen fibrils. CONCLUSION: In stage of the primary frozen shoulder exclusive fibrosis of the joint capsule was found. Electronmicroscopic twists of collagen fibrills were marked.

[Subjective and clinical outcome after rotator cuff repair in elderly patients]. / Subjektives und klinisches Outcome nach Rekonstruktion der Rotatorenmanschette im höheren Lebensalter.

AIM: The indication for rotator cuff repair in elderly patients is controversial. Results after refixation of the cuff in dependence on age, size of rupture and retraction using the Constant-Murley and DASH scores were analysed. We wanted to examine whether the results of rotator cuff repair allow us to define the indication for the operative procedure. METHODS: Over a period of six years 154 patients with symptomatic rotator cuff lesion had undergone repair of cuff tears, and 119 patients were evaluated for follow-up applying the Constant-Murley and DASH scores for self-assessment. RESULTS: On average a postoperative Constant-Murley score of 83.5 points was obtained. A significant difference between the operated and the opposite shoulder was observed. In the DASH score a mean point value of 23.6 was calculated. In all combined groups more than 50% of the patients and 27% of the patients in the Bateman IV group were satisfied with the postoperative results using both scores. Reruptures were seen mostly in the Bateman IV group. CONCLUSION: The difference of score points between the operated to the opposite site in Bateman II and III groups did not show unfavourable values at higher age whereas adverse data were found in the Bateman IV group. In the Bateman IV group, in addition to cuff repair a muscle flap or arthroplasty should be considered in the choice of operative procedure.

[Pseudoaneurysm - a rare complication after insertion of a distal locking bolt of an intramedullary femur nail]. / Das Aneurysma spurium - eine seltene Komplikation nach distaler Verriegelung eines Femurmarknagels.

A pseudoaneurysm is caused by an extravasal hematoma after vessel injury. Persistent blood supply via the afferent artery can lead to aneurysm growth with potential rupture of the lesion. We present a case of a 27-year-old man who developed three independent pseudoaneurysms after insertion of percutaneous distal locking bolts for static locking of an intramedullary femur nail. Color-coded Doppler sonography and intraarterial subtraction angiography were used to detect the lesion. Successful treatment consisted of endovascular embolization (coiling) with injection of a platinum coil into the aneurysm's supplying vessel. Pseudoaneurysm after fracture or fracture fixation represents a rare complication, but the risk of acute bleeding by pseudoaneurysm rupture should not be neglected.

mRNA expression and protein distribution of fibronectin splice variants and high-molecular weight tenascin-C in different phases of human fracture healing.

Fracture healing is a reparative physiological process, which proceeds in stages, each characterized by the predominant tissue in the fracture gap. The tissue matrix is continuously reorganized by cell migration, proliferation, and differentiation. Adhesive proteins such as fibronectin and tenascin transmit information between matrix and cells. As a result of alternative splicing of pre-RNA, EDA + fibronectin, EDB + fibronectin, and high-molecular weight (hm) tenascin-C are generated. By definition, EDB + fibronectin is an oncofetal protein because it is extremely rare in normal adult tissue and plasma, whereas it is expressed in fetal and tumor tissues and during wound healing. In this study, we for the first time describe EDA + fibronectin, EDB + fibronectin, and hm tenascin-C expression in human fracture gap tissue during various stages of differentiation. We demonstrate mRNA expression of all three splice variants in the initial fibrin matrix with upregulation in the enchondral ossification/osteoid and woven bone stages. Of all variants, EDA + fibronectin mRNA has the highest concentration in all stages. For the analysis, we used LightCycler-based relative mRNA quantification and immunohistochemistry. Our data demonstrate that EDA + fibronectin and hm tenascin-C show a diffuse distribution pattern in fracture gap connective tissue, while EDB + fibronectin is focally concentrated in osteoblastic cells at the margins of woven bone. EDA + fibronectin and hm tenascin represent markers for active granulation processes, whereas EDB + fibronectin is specific for cells forming the enchondral and osteoid matrix. The possibility of stimulating fracture healing by EDB + fibronectin-cytokine complexes should be tested in further investigations.

[Elution kinetics and antimicrobial effects of gentamicin- and clindamycin-loaded bone cements in vitro]. / In-vitro-Untersuchungen zu Elutionskinetiken und antimikrobiellen Wirkungen von Gentamicin- und Clindamycin-haltigen Knochenzementen.

AIMS: In arthroplasty and particulary in revision surgery antibiotic-loaded bone cements are applied. The purpose of the current study was to analyse the kinetics of antibiotic release and antimicrobial effects of the gentamicin- and clindamycin-loaded bone cement Versabond. MATERIALS AND METHODS: Test cylinders of bone cements, containing gentamicin and clindamycin in different concentrations, were investigated in vitro with regards to the kinetics of antibiotic release by applying static and continuous elution methods. Antimicrobial effects of antibiotic-loaded bone cement were tested in an infection model with different strains of Staphylococcus aureus and Staphylococcus epidermidis and live/dead dye plus fluorescence microscopy. For the surface analysis of antibiotic-loaded test cylinders contaminated with Staphylococcus epidermidis, scanning electron microscopy was used. RESULTS: The measurement of static and continuous elution revealed initially higher rates of gentamicin release in comparison with clindamycin. The gentamicin amount decreased within 24 h, whereas clindamycin-loaded test cylinders showed a prolonged release. After 24 h exclusive avital bacteria on antibiotic-loaded cement cylinders were seen, after 72 h on gentamicin-loaded cement bacterial growth was again detected. Application of antibiotics in bone cement revealed an inhomogeneous surface of the bone cement cylinder. CONCLUSIONS: On application of clindamycin in bone cement the features of increased antibiotic uptake and antimicrobial effect compared with gentamicin-loaded bone cement are observed.

[Biochemical bone resorption markers during the healing of osteoporotic fractures]. / Biochemische Knochenresorptionsmarker in der Heilungsphase osteoporotischer Frakturen.

BACKGROUND: This study investigated the progression and clinical relevance of biochemical resorption marker values during fracture healing in osteoporosis. PATIENTS AND METHODS: In 44 patients with distal radius fractures and 29 patients without fractures, the blood and urine concentrations of pyridinoline (PYD), deoxypyridinoline (DPD), N-telopeptides (NTx), and bone sialoprotein (BSP) were recorded on the day of trauma as well as during further progression. All postmenopausal patients underwent bone density measurement. Accordingly, patients were divided into premenopausal, postmenopausal osteoporotic, and postmenopausal nonosteoporotic groups. RESULTS: Between the groups, PYD, DPD, and NTx showed significant differences in their initial values. However, their further relative progression was primarily affected by the chosen therapy. CONCLUSION: Bone resorption markers can diagnostically point to osteoporosis and are significant parameters in fracture healing.

[Covering of soft tissue defects of the lower leg with adipo-facial flaps]. / Neurovaskuläre Lappenplastiken zum Verschluss problematischer Weichteildefekte am Unterschenkel.

AIM OF THE STUDY: Soft tissue covering on the lower leg is a difficult challenge. A plastic surgical method for covering of these soft tissue defects is presented. PATIENTS AND METHODS: From May 1997 until May 2003 36 patients were treated using neurovascular flaps. Above the sural or the saphenous nerve an adipo-facial flap is dissected, rotated into the defect and fixed without tension. Wound closure of the donor-site defect is done primarily or by mesh-graft. RESULTS: In 35 cases the flap healed without functional impairment. Primary healing was achieved in 30 patients. In 5 cases partial loss of the skin island was registered, whereby subcutaneous tissue remained vital. Revision by mesh-graft transplantation led to successful healing in these patients. In only one patient necrosis of the flap was seen. CONCLUSION: Neurovascular flaps usually result in reliable and complete healing of soft tissue defects of the lower extremity.

[Visual test evaluation of the results for the semiquantitative determination of cystine in urine using the nickel/dithionite (Ni2+/S2O42-) test]. / Visuelle Auswertung von Analyseergebnissen zur semiquantitativen Cystinbestimmung im Urin mit dem Nickel/Dithionit (Ni2+/S2O4(2-))-Test.

The Ni2+/S2O4(2-)-test makes semiquantitative assessment of cystine concentration in urine possible. The reaction principle of the method is based on the formation of a nickel/cysteine/sulfide complex of brown colour. Visual test evaluation is done by classifying the colour intensity of the sample/reagent mixture according to an exact colour shade. Each colour shade on a colour reference table is correlated to a certain range of cystine concentration. Cystine determination is described in detail for two ranges (0.60 and 1.25 mmol/l).

[A semiquantitative rapid test for cysteine: possible use for monitoring the ascorbic acid therapy in cystinuria and cystine lithiasis]. / Semiquantitativer Cystein-Schnelltest Möglichkeit zur Verlaufskontrolle der Ascorbinsäuretherapie bei Cystinurie und Cystinlithiasis.

A semiquantitative method is presented for the determination of cysteine in urine, based on the formation of a red cysteine/nitroprusside salt. The method is suitable as a rapid test for checking the progress of ascorbic acid therapy of cystinuria and cystine urolithiasis. It guarantees acceptable reproducibility of values and can be easily carried out in any clinical chemical laboratory. With the K2CO3/nitroprusside test described and an additional colorimetric determination of cystine (CN-/nitroprusside), a separate semiquantitative differentiation of cysteine and cystine in fresh (!) urine is possible.

[Simple rapid test with carbonate/nitroprusside for the semiquantitative determination of cysteine in the urine--possible use for control of ascorbic acid therapy in cystine urolithiasis]. / Einfacher Schnelltest mit Karbonat/Nitroprussid zur semiquantitativen Bestimmung von Zystein im Urin--Möglichkeit einer Verlaufskontrolle unter Askorbinsäuretherapie beim Zystinsteinleiden.

In this paper a method of semiquantitative cysteine determination is presented, which is based on the formation of a red cysteine-Na-nitroprusside salt. The method is a suitable rapid test for checking the process of ascorbic acid therapy in cystinuria and cystine urolithiasis patients. It guarantees acceptable reproducibility of values and can be easily carried out in every clinicochemical laboratory. With the K2CO3/nitroprusside test described and an additional cystine rapid test (Ni2+/S2O4(2-) tablet reagency) a separate semiquantitative differentiation of cysteine and cystine in fresh (!) urine is possible.

Investigations on the early diagnosis of cystinuria in children.

The procedure described confirms that the Ni2+/S2O4(2-) tablet test is a suitable quick cystine screening test for large groups. In 16 (0.67%) of 2,395 one- to three-year-old crèche children significantly enhanced cystine levels of 0.60-0.96 mmol/l were found. The corresponding creatinine values (mmol/l) did not reveal any significant deviations from the normal values. In a preliminary group of 9 children, enhanced lysine levels support the suspicion of the existence of cystinuria without clinical symptoms. This also forms the basis for further necessary family histories as well as for prophylactic measures.

[Modified rapid test for the semiquantitative determination of cystine in the urine]. / Modifizierter Schnelltest zur semiquantitativen Bestimmung von Cystin im Urin.

The present study describes a modified method of the semiquantitative determination of cystine in the urine which is based on the decomposition of cystine by dithionite ions and formation of a mixed nickel/cystein/sulfide-complex. The method is suitable as rapid and search test, it guarantees an acceptable reproducibility as well as the exactness of the values and is simply to be performed in every clinical-chemical laboratory. The criteria of analytic applicability are confirmed in comparisons of methods. It must be urged to treat fresh urines.

[Difficulties involved in the Hoffa fractures]. / Die Problematik der Hoffa-Fraktur.

To study the outcome in Hoffa fractures regarding the type of fracture, concomitant injuries, and different treatments. From January 1996 until December 2002, 7 Hoffa fractures (6 patients) from a total of 79 distal femoral fractures were treated in the Department of Trauma Surgery, University Hospital of Giessen. The results were evaluated by Neer's score. For the evaluation of postoperative vascularization of the distal femur, clinical and radiological findings were compared with anatomical studies of the bone arteries by corrosion preparations. Fracture consolidation was seen in all cases. Patients with an isolated Hoffa fracture achieved a score of 89.0 (82-97) according to Neer. One patient suffering initially from nonunion achieved 68 points after bone union. One polytraumatized patient with bilateral Hoffa fracture achieved a satisfying result (score value of 72) and a bad result (score value of 38). Multiple small nutrient arteries of the distal femur originate from the "rete articulare genus." They can be expected to ensure the arterial blood supply. Hoffa fracture is a very rare injury. In our patients, careful surgical treatment with open reduction and safe screw fixation yielded good to very good results.