Molecular recognition of structurally disordered Pro/Ala-rich sequences (PAS) by antibodies involves an Ala residue at the hot spot of the epitope.

Pro/Ala-rich sequences (PAS) are polypeptides that were developed as a biological alternative to poly-ethylene glycol (PEG) to generate biopharmaceuticals with extended plasma half-life. Like PEG, PAS polypeptides are conformationally disordered and show high solubility in water. Devoid of any charged or prominent hydrophobic side chains, these biosynthetic polymers represent an extreme case of intrinsically disordered proteins. Despite lack of immunogenicity of PAS tags in numerous animal studies we now succeeded in generating monoclonal antibodies (MAbs) against three different PAS versions. To this end, mice were immunized with a PAS#1, P/A#1 or APSA 40mer peptide conjugated to keyhole limpet hemocyanin as highly immunogenic carrier protein. In each case, one MAb with high binding activity and specificity towards a particular PAS motif was obtained. The apparent affinity was strongly dependent on the avidity effect and most pronounced for the bivalent MAb when interacting with a long PAS repeat. X-ray structural analysis of four representative anti-PAS Fab fragments in complex with their cognate PAS epitope peptides revealed interactions dominated by hydrogen bond networks involving the peptide backbone as well as multiple Van der Waals contacts arising from intimate shape complementarity. Surprisingly, Ala, the L-amino acid with the smallest side chain, emerged as a crucial feature for epitope recognition, contributing specific contacts at the center of the paratope in several anti-PAS complexes. Apart from these insights into how antibodies can recognize feature-less peptides without secondary structure, the MAbs characterized in this study offer valuable reagents for the preclinical and clinical development of PASylated biologics.

Periarticular injections with continuous perfusion of local anaesthetics provide better pain relief and better function compared to femoral and sciatic blocks after TKA: a randomized clinical trial.

PURPOSE: Combined femoral and sciatic nerve blocks for post-operative pain management following total knee arthroplasty (TKA) improve patient satisfaction, decrease narcotic consumption and improve pain. However, accompanying motoric weakness can cause falls and related complications. We wonder whether peri-capsular injections in combination with intra-articular perfusion of local anaesthetics would result in equal or less pain without the related complications of nerve blocks. The objective of the study was to verify these aspects in a prospective randomized trial comparing both treatments. METHODS: Fifty TKA patients randomly received either a femoral (continuous) and a sciatic (single-shot) nerve block (CFNB group, 25 knees) or periarticular infiltrations and a continuous post-operative intra-articular infusion (PIAC group, 25 knees). VAS for pain, pain medication consumption, functional assessment, straight leg raising as well as KSS were recorded post-operatively for 6 days. RESULTS: VAS (p < 0.001) and KSS (p = 0.05) were significantly better for PIAC. There was increased pain following CFNB compared to PIAC. Catheters stayed for 4 days, a pain 'rebound' occurred after removing in CFNB but not after PIAC. There was no difference in regard to knee function (n.s.), but straight leg raising was significant better following PIAC. There were two falls in patients with CFNB. CONCLUSION: Peri-capsular injections combined with an intra-articular catheter provide better pain control, no rebound pain with better function and might decrease the risk of complications related to motor weakness. LEVEL OF EVIDENCE: I.

[Partial replacement of the knee joint with patient-specific instruments and implants (ConforMIS iUni, iDuo)]. / Patientenspezifische Instrumente und Implantate beim Teilgelenkersatz des Kniegelenkes (ConforMIS iUni, iDuo).

Knee arthroplasty is a successful standard procedure in orthopedic surgery; however, approximately 20 % of patients are dissatisfied with the clinical results as they suffer pain and can no longer achieve the presurgery level of activity. According to the literature the reasons are inexact fitting of the prosthesis or too few anatomically formed implants resulting in less physiological kinematics of the knee joint. Reducing the number of dissatisfied patients and the corresponding number of revisions is an important goal considering the increasing need for artificial joints. In this context, patient-specific knee implants are an obvious alternative to conventional implants. For the first time implants are now matched to the individual bone and not vice versa to achieve the best possible individual situation and geometry and more structures (e.g. ligaments and bone) are preserved or only those structures are replaced which were actually destroyed by arthrosis. According to the authors view, this represents an optimal and pioneering addition to conventional implants. Patient-specific implants and the instruments needed for correct alignment and fitting can be manufactured by virtual 3D reconstruction and 3D printing based on computed tomography (CT) scans. The portfolio covers medial as well as lateral unicondylar implants, medial as well as lateral bicompartmental implants (femorotibial and patellofemoral compartments) and cruciate ligament-preserving as well as cruciate ligament-substituting total knee replacements; however, it must be explicitly emphasized that the literature is sparse and no long-term data are available.

Effective prevention of recurrent dislocation following primary cemented Endo-MarkIII/SP2 total hip arthroplasty using a posterior lip augmentation device.

PURPOSE: This retrospective study was performed to determine the effectiveness of preventing recurrent dislocation following primary cemented Endo-MarkIII/SP2 total hip replacement using a posterior lip augmentation device (PLAD). METHODS: Between January 2003 and Dezember 2006, 27 PLADs were used in the treatment of recurrent hip dislocation in 27 patients who had received a cemented primary total hip arthroplasty using Endo-MarkIII/SP2 (Waldemar LINK, Hamburg, Germany) components. The mean number of dislocations prior to stabilization with this specific device was 2.6 (range 2-4, SD ± 0.4) with a mean time to revision surgery of 10 months (IQR 13). The mean age of the patients at time of revision surgery was 81.5 years (range 70-94, SD ± 6.9). The control group evaluating the clinical outcome using the Harris Hip Score (HHS) also received a cemented primary total hip arthroplasty using the same implants. A retrospective clinical and radiological review was carried out at a mean follow-up of 68.5 months (range 30-103, SD ± 17.7). RESULTS: Of the 27 patients, 6 had died at the time of the latest review, with the posterior lip augmentation device still in situ and without reported further dislocation after PLAD application. In 2 of the remaining 21 patients recurrent dislocation occurred, thus a subsequent revision of respective implants had to be performed to achieve persistent joint stability. At latest follow-up no deep infection or implant loosening occurred. CONCLUSION: Surgical treatment of recurrent dislocation following primary cemented Endo-MarkIII/SP2 total hip replacement using a posterior lip augmentation device is a safe and effective procedure which can lead to a secondary stabilization of the total hip arthroplasty in about 90 % of the patients.

Increased risk for extended acetabular reconstruction in failed hip resurfacing as compared to failed total hip arthroplasty.

INTRODUCTION: Hip resurfacing (HR) is intended to preserve the femoral bone stock during primary arthroplasty. On the other hand, little has been reported regarding the intraoperative need of bone reconstruction for extended acetabular defects during hip resurfacing revision. Thus, the aim of the presented study was to identify whether there is an increased need for acetabular bone reconstruction in HR revision surgery. MATERIALS AND METHODS: We analyzed the data of 38 patients who underwent 39 conversions from a HR to a total hip arthroplasty (THA). Acetabular bone defects and the respective revision technique were compared against a temporary cohort of patients undergoing revision surgery of a conventional THA. RESULTS: In 29 HR patients revision required either autogenous or allogenous impaction bone grafting to adequately manage acetabular host bone degradation. In 10 cases additional implantation of a reinforcement device was necessary. Compared to the THA cohort revision of failed HR is associated with a significantly increased risk of higher grade bone defects (Paprosky classification) and extended acetabular reconstruction (p < 0.05). CONCLUSIONS: This study provides evidence that revision of failed HR devices is associated with an increased risk for extensive acetabular defects. Furthermore, the preoperative radiographic assessment of HR devices often underestimates the intraoperative acetabular defect. Surgeons should be aware of this fact not to technically underestimate HR revision procedures.

The Singh Index does not correlate with bone mineral density (BMD) measured with dual energy X-ray absorptiometry (DXA) or peripheral quantitative computed tomography (pQCT).

The Singh Index (SI), a classification system by which the severity of osteoporosis is assessed based on plain radiographs, is a renowned, simple and inexpensive form of evaluating osteoporosis. The aim of this study was to evaluate the correlation between the SI and bone mineral density (BMD) as measured by dual energy X-ray absorptiometry (DXA) and peripheral quantitative computed tomography (pQCT). The SI was evaluated in 128 cadaveric femora (64 patients, mean age 66.7 years, range 24-89 years) by three independent observers, all blinded to plain radiographs. BMD was also analysed by means of DXA and pQCT in the cadaveric femora. The mean interrater correlation was found to be 0.629. The correlation of the mean BMD measured by DXA (DXA-BMD) and SI was found to be poor, with r = 0.49. The corresponding sensitivity of 45.2 % and specificity of 92.3 % were even poor. The BMD measured by pQCT (pQCT-BMD) also revealed a poor correlation with SI, such that r = 0.337 and r = 0.428 for the trochanteric and neck regions, respectively. Due to the poor correlation of the SI with BMD and the poorer interrater correlation, the SI should be rejected as a tool for evaluating osteoporosis. The SI was found to be too imprecise and is therefore unsuitable for diagnosing osteoporosis and osteopenia.

[The use of tantalum cones for reconstruction of bone defects in revision total knee arthroplasty]. / Defektrekonstruktion beim Knieendoprothesenwechsel mit Tantal-Konen.

OBJECTIVE: Revision arthroplasty of the knee is often associated with substantial femoral and/or tibial bone loss. Tantalum cones are used to reconstruct these defects and to improve initial stability. This requires an implantation in the "press-fit" technique with maximum contact to the host bone. INDICATIONS: Tantalum cones may be used in grade 2-3 femoral and/or tibial defects according to the AORI (Anderson Orthopedic Research Institute) classification system. CONTRAINDICATIONS: There are no contraindications described. SURGICAL TECHNIQUE: After removal of the implant and cement remnants, bone defects have to be evaluated. A tantalum cone which adequately fills the bone defect is implanted using the "press-fit" technique. If necessary, saving resection of surplus bone to fit the cone properly. Gaps between the cone and the host bone are filled with cancellous bone in "impaction-bone-grafting" technique to increase the area of contact. Fitting the revision knee prosthesis and fixing with the use of bone cement. POSTOPERATIVE MANAGEMENT: Postoperative physiotherapy is adjusted to the result of the reconstruction. In most cases with stable reconstruction, mobilization with full weight-bearing and the use of two crutches can be performed. Additional bone grafting may require a partial weight-bearing regimen for postoperative mobilization. Physiotherapy to improve range of motion is performed starting on postoperative day 1. RESULTS: Several studies reported promising midterm results (observation period about 36 months) after implantation of tantalum cones in revision knee arthroplasty. There is consistent evidence for stable osteointegration of the cones. The main intraoperative complication is fracture of the host bone during impaction of the cones.

Single-stage revision for fungal peri-prosthetic joint infection: a single-centre experience.

Fungal peri-prosthetic infections of the knee and hip are rare but likely to result in devastating complications. In this study we evaluated the results of their management using a single-stage exchange technique. Between 2001 and 2011, 14 patients (ten hips, four knees) were treated for a peri-prosthetic fungal infection. One patient was excluded because revision surgery was not possible owing to a large acetabular defect. One patient developed a further infection two months post-operatively and was excluded from the analysis. Two patients died of unrelated causes. After a mean of seven years (3 to 11) a total of ten patients were available for follow-up. One patient, undergoing revision replacement of the hip, had a post-operative dislocation. Another patient, undergoing revision replacement of the knee, developed a wound infection and required revision 29 months post-operatively following a peri-prosthetic femoral fracture. The mean Harris hip score increased to 74 points (63 to 84; p < 0.02) in those undergoing revision replacement of the hip, and the mean Hospital for Special Surgery knee score increased to 75 points (70 to 80; p < 0.01) in those undergoing revision replacement of the knee. A single-stage revision following fungal peri-prosthetic infection is feasible, with an acceptable rate of a satisfactory outcome.

Femoral stem impaction grafting: extending the role of cement.

Femoral revision after cemented total hip replacement (THR) might include technical difficulties, following essential cement removal, which might lead to further loss of bone and consequently inadequate fixation of the subsequent revision stem. Femoral impaction allografting has been widely used in revision surgery for the acetabulum, and subsequently for the femur. In combination with a primary cemented stem, impaction grafting allows for femoral bone restoration through incorporation and remodelling of the impacted morsellized bone graft by the host skeleton. Cavitary bone defects affecting meta-physis and diaphysis leading to a wide femoral shaft, are ideal indications for this technique. Cancellous allograft bone chips of 1 mm to 2 mm size are used, and tapered into the canal with rods of increasing diameters. To impact the bone chips into the femoral canal a prosthesis dummy of the same dimensions of the definitive cemented stem is driven into the femur to ensure that the chips are very firmly impacted. Finally, a standard stem is cemented into the neo-medullary canal using bone cement. To date several studies have shown favourable results with this technique, with some excellent long-term results reported in independent clinical centres worldwide.

Infection rates in patients undergoing primary knee arthroplasty with pre-existing orthopaedic fixation-devices.

BACKGROUND: Prior knee surgery in the setting of knee arthroplasty (KA) can influence the overall outcome of the procedure and render the operation more technically challenging. The effects of residual fixation devices on subsequent procedures about the knee are ill-defined. Some authors claim an increase in periprosthetic infection in this cohort of patients. The objective of this study was to evaluate the overall incidence of periprosthetic infections in patients undergoing primary KA with pre-existing osteosynthetic hardware in situ. METHODS: The current investigators retrospectively reviewed 124 patients undergoing knee arthroplasty and removal of orthopaedic fixation devices, due to prior high tibial osteotomies, fracture fixation or cruciate ligament reconstruction. The exclusion criterion was a prior history of infection of the fixation device. The mean follow-up time was 5.4 years (range 15 months to 9 years). Nine patients were lost to follow-up. RESULTS: Joint aspiration was performed two weeks prior to surgery in 53 patients (42.4%) and intra-operative samples were obtained in 106 patients (84.8%), which did not show any bacterial growth. A subacute periprosthetic infection occurred after seven months in only one patient. CONCLUSION: The results of the current study demonstrate that previously implanted osteosynthetic fixation devices do not significantly increase the risk of developing periprosthetic knee infections. A two-stage procedure with implant retrieval prior to total knee arthroplasty is not clinically indicated in the cohort described, amongst whom an infection rate of 0.9% was revealed.

[Revision of unconstrained total knee arthroplasty - a technical analysis]. / Oberflächenersatzwechsel in der Knieendoprothetik - eine intraoperative Ursachenanalyse.

BACKGROUND: Total knee arthroplasty (TKA) revisions are increasing due to the rising numbers of primary cases, younger patients and an aging population. The technical demand of these procedures is incomparable higher than in primary cases. Also the expectable clinical results are inferior. Measures to improve the clinical results are desireable. PATIENTS/MATERIAL: 203 unconstrained knee arthroplasties were explanted in 2009 in a single centre. In 150 cases those were TKAs. Fractures were excluded. These cases were analysed with special attention to the reason for the failure. Clinical and radiological data were included. RESULTS: Survival of revised implants was in the mean 55 months (range: 0,5-125 months). In 46 cases components showed an aseptic loosening (30.7 %), 37 TKAs were unstable (24.7 %), in 26 cases stiffness was detected (17.3 %), and nine misplaced TKA components (five femoral, two tibial, two combined; total 6 %) as well as three broken inlays (2 %) were encountered. In 25 cases deep infection was observed (16.7 %) and four patients complained of an unspecific anterior knee pain (2.6 %). Three TKAs were removed for a two-staged procedure. In five cases an inlay exchange was performed and in another five cases a conversion from cruciate retaining to posterior stabilised state was performed. In 60 cases a rotating hinged TKA was implanted (40 %) and in 42 a pure hinge (28 %). In 45 cases a condylar constrained TKA system (CCK, 30 %) was used. Range of movement increased with the procedure. Plain radiographs were inferior in detecting all loosening in advance. CONCLUSION: This study demonstrated that in more than one third of the cases the revision could have been avoided and was due to technical errors during the primary procedure. Infection and arthrofibrosis were besides unspecific complaints less often observed than is described in the literature. This study supports the high demand on the technical issues during the primary procedure which should be conducted by experienced surgeons. Registry data from Germany should allow the provision of more specific information in the future.

Fracture prevention by femoroplasty--cement augmentation of the proximal femur.

The prevention of hip fractures is a desirable goal to reduce morbidity, mortality, and socio-economic burden. We evaluated the influence on femoral strength of different clinically applicable cementing techniques as "femoroplasty." Twenty-eight human cadaveric femora were augmented by means of four clinically applicable percutaneous cementing techniques and then tested biomechanically against their native contralateral control to determine fracture strength in an established biomechanical model mimicking a fall on the greater trochanter. The energy applied until fracture could be significantly increased by two of the methods by 160% (53.1 Nm vs. 20.4 Nm, p < 0.001) and 164% (47.1 Nm vs. 17.8 Nm, p = 0.008), respectively. The peak load to failure was significantly increased by three of the methods by 23% (3818.3 N vs. 3095.7 N, p = 0.003), 35% (3698.4 N vs. 2737.5 N, p = 0.007), and 12% (3056.8 N vs. 2742.8 N, p = 0.005), respectively. The femora augmented with cemented double drill holes had a lower fracture strength than the single drilled ones. Experimental femoroplasty is a technically feasible procedure for the prophylactic reinforcement of the osteoporotic proximal femur and, hence, could be an auxiliary treatment option to protect the proximal femur against osteoporotic fractures.

Skeletal mineralization defects in adult hypophosphatasia--a clinical and histological analysis.

UNLABELLED: Histomorphometry and quantitative backscattered electron microscopy of iliac crest biopsies from patients with adult hypophosphatasia not only confirmed the expected enrichment of non-mineralized osteoid, but also demonstrated an altered trabecular microarchitecture, an increased number of osteoblasts, and an impaired calcium distribution within the mineralized bone matrix. INTRODUCTION: Adult hypophosphatasia is an inherited disorder of bone metabolism caused by inactivating mutations of the ALPL gene, encoding tissue non-specific alkaline phosphatase. While it is commonly accepted that the increased fracture risk of the patients is the consequence of osteomalacia, there are only few studies describing a complete histomorphometric analysis of bone biopsies from affected individuals. Therefore, we analyzed iliac crest biopsies from eight patients and set them in direct comparison to biopsies from healthy donors or from individuals with other types of osteomalacia. METHODS: Histomorphometric analysis was performed on non-decalcified sections stained either after von Kossa/van Gieson or with toluidine blue. Bone mineral density distribution was quantified by backscattered electron microscopy. RESULTS: Besides the well-documented enrichment of non-mineralized bone matrix in individuals suffering from adult hypophosphatasia, our histomorphometric analysis revealed alterations of the trabecular microarchitecture and an increased number of osteoblasts compared to healthy controls or to individuals with other types of osteomalacia. Moreover, the analysis of the mineralized bone matrix revealed significantly decreased calcium content in patients with adult hypophosphatasia. CONCLUSIONS: Taken together, our data show that adult hypophosphatasia does not solely result in an enrichment of osteoid, but also in a considerable degradation of bone quality, which might contribute to the increased fracture risk of the affected individuals.

Preproenkephalin (Penk) is expressed in differentiated osteoblasts, and its deletion in Hyp mice partially rescues their bone mineralization defect.

Although our understanding of the molecular mechanisms controlling osteoblast differentiation and function is steadily increasing, there are still many open questions, especially regarding the regulation of bone matrix mineralization. For instance, while there is hallmark evidence for the importance of the endopeptidase Phex, whose inactivation in Hyp mice or human patients causes X-linked hypophosphatemic rickets, it is still largely unknown how Phex controls bone mineralization since a physiological substrate for its endopeptidase activity has not been identified yet. Using a genome-wide expression analysis comparing primary calvarial osteoblasts, we have identified preproenkephalin (Penk) as a gene that is selectively expressed in mineralized cultures. Since a role of enkephalin in the regulation of bone remodeling has been suggested previously and since Leu-enkephalin is known to be cleaved by Phex, we analyzed whether Penk expression in osteoblasts is physiologically relevant. Through skeletal analysis of a Penk-deficient mouse model, we found that Penk expression is dispensable for bone development and remodeling since we could not detect any defect following nondecalcified bone histology and histomorphometry compared to wild-type littermates. When Penk was deleted in Phex-deficient Hyp mice, however, we observed a significant reduction of the osteoid enrichment at 24 weeks of age, whereas their disturbance of mineral homeostasis was not affected by the additional absence of the Penk gene. Taken together, our data provide the first in vivo analysis concerning the role of Penk in osteoblasts.

The treatment of intertrochanteric fractures: results using an intramedullary nail with integrated cephalocervical screws and linear compression.

OBJECTIVE: A new device for the treatment of intertrochanteric fractures that uses 2 cephalocervical screws in an integrated mechanism allowing linear intraoperative compression and rotational stability of the head/neck fragment has been developed. The aim of this study was to describe the results using this device for the treatment of stable and unstable intertrochanteric fractures. DESIGN: Prospective, consecutive. SETTING: Academic Trauma Center. METHODS: Between March 1, 2005, and July 31, 2006, 100 consecutive patients with an intertrochanteric fracture were treated with a new trochanteric antegrade nail (InterTan; Smith-Nephew, Memphis, TN). All living patients were followed up for a minimum of 1 year postoperatively (range 12-27 months). Clinical and radiographic examinations were performed until healing and at the 1-year anniversary of the index procedure. Healing, pain with ambulation, return to activities of daily living, the modified Harris hip score, and Barthel Index were used to evaluate outcomes. RESULTS: The mean age of the patients was 81.2 (+/-11.3) years. Thirty-seven patients died, 12 were too infirmed for follow-up, and 3 could not be located, leaving 48 patients available for final evaluation. The average surgical time was 41 minutes (13-95 minutes). This rose significantly with the complexity of the fracture (OTA/AO classification: A1 versus A3, P = 0.016). All fractures healed within 16 weeks (range 10-16 weeks). Radiographic analysis at healing revealed no loss of reduction, no uncontrolled collapse of the neck, no nonunions, no femoral shaft fractures, and no implant failures. Two cases in the series were poorly reduced and settled into varus malalignment. There was no varus malposition seen in the remaining 46 fractures. The mean prefracture Harris hip score (75.1 +/- 13.4) was significantly reduced at the time of follow-up (70.3 +/- 14.5, P = 0.003); 58% of the patients recovered their prefracture status. No significant difference was seen for the Barthel Index. CONCLUSIONS: The InterTan device appears to be a reliable implant for the treatment of intertrochanteric femoral fractures. Its design provides for stability against rotation and minimizes neck malunions (shortening) through linear intraoperative compression of the head/neck segment to the shaft. As a result of the negligible complication rate and improved clinical outcomes, this implant is now the standard treatment for all intertrochanteric fractures at our institution.

[Periprosthetic fractures. Long-term results after plate osteosynthesis stabilization]. / Periprothetische Femurfrakturen. Langzeitergebnisse nach plattenosteosynthetischer Stabilisierung.

BACKGROUND: Due to the increasing age of patients and the rising number of joint replacements, the incidence of periprosthetic fractures (PPF) is also increasing. The treatment should be selected with knowledge of the prefracture interface status and the type of fracture involved. The aim of this study was to evaluate our treatment of PPF with long-term follow-up. PATIENTS AND METHODS: From 1988 to 2006, 99 patients with PPF were treated in our department. In 86 cases a plate osteosynthesis was used. After a mean time of 7.3+/-2.8 years, we studied 56 patients and monitored their complications. RESULTS: The most diagnosed fracture was Johansson type III (44%). Seventy-one patients were treated with a conventional and 15 with locking-plate osteosynthesis. In 15 cases (17.5%) we found severe complications (3 breaks and 3 dislocations of the plates, 6 cases of pseudarthrosis, 2 deep wound infections, and 1 case of postoperative bleeding). CONCLUSION: Due to the minor frequency of severe complications, plate osteosynthesis of a periprosthetic fracture with a loosened interface is a good therapeutic option for individual patients, particularly for geriatric patients and those without disorders specific to a loosened interface.

Phylogeny and ecophysiology of opportunistic "snow molds" from a subalpine forest ecosystem.

Mats of coenocytic "snow molds" are commonly observed covering the soil and litter of alpine and subalpine areas immediately following snow melt. Here, we describe the phylogenetic placement, growth rates, and metabolic potential of cold-adapted fungi from under-snow mats in the subalpine forests of Colorado. SSU rDNA sequencing revealed that these fungi belong to the zygomycete orders Mucorales and Mortierellales. All of the isolates could grow at temperatures observed under the snow at our sites (0 degrees C and -2 degrees C) but were unable to grow at temperatures above 25 degrees C and were unable to grow anaerobically. Growth rates for these fungi were very high at -2 degrees C, approximately an order of magnitude faster than previously studied cold-tolerant fungi from Antarctic soils. Given the rapid aerobic growth of these fungi at low temperatures, we propose that they are uniquely adapted to take advantage of the flush of nutrient that occurs at the soil-snow interface beneath late winter snow packs. In addition, extracellular enzyme production was relatively high for the Mucorales, but quite low for the Mortierellales, perhaps indicating some niche separation between these fungi beneath the late winter snow pack.

Gap junctional communication in human osteoclasts in vitro and in vivo.

Bone-forming cells are known to be coupled by gap junctions, formed primarily by connexin43 (Cx43). The role of Cx43 in osteoclasts has so far only been studied in rodents, where Cx43 is important for fusion of mononuclear precursors to osteoclasts. Given the potential importance for human diseases with pathologically altered osteoclasts, we asked whether a similar influence of Cx43 can also be observed in osteoclasts of human origin. For this purpose, Cx43 mRNA expression was studied in a time course experiment of human osteoclast differentiation by RT-PCR. Localization of Cx43 in these cells was determined by immunohistochemistry and confocal microscopy. For the assessment of the effect of gap junction inhibition on cell fusion, gap junctions were blocked with heptanol during differentiation of the cells and the cells were then evaluated for multinuclearity. Paraffin sections of healthy bone and bone from patients with Paget's disease and giant cell tumour of the bone were used to study Cx43 expression in vivo. We found mRNA and protein expression of Cx43 in fully differentiated osteoclasts as well as in precursor cells. This expression decreased in the course of differentiation. Consistently, we found a lower expression of Cx43 in osteoclasts than in bone marrow precursor cells in the histology of healthy human bone. Blockade of gap junctional communication by heptanol led to a dose-dependent decrease in multinuclearity, suggesting that gap junctional communication precedes cell fusion of human osteoclasts. Indeed, we found a particularly strong expression of Cx43 in the giant osteoclasts of patients with Paget's disease and giant cell tumour of the bone. These results show that gap junctional communication is important for fusion of human mononuclear precursor cells to osteoclasts and that gap junctional Cx43 might play a role in the regulation of size and multinuclearity of human osteoclasts in vivo.

The protein tyrosine phosphatase Rptpzeta is expressed in differentiated osteoblasts and affects bone formation in mice.

Tyrosine phosphorylation of intracellular substrates is one mechanism to regulate cellular proliferation and differentiation. Protein tyrosine phosphatases (PTPs) act by dephosphorylation of substrates and thereby counteract the activity of tyrosine kinases. Few PTPs have been suggested to play a role in bone remodeling, one of them being Rptpzeta, since it has been shown to be suppressed by pleiotrophin, a heparin-binding molecule affecting bone formation, when over-expressed in transgenic mice. In a genome-wide expression analysis approach we found that Ptprz1, the gene encoding Rptpzeta, is strongly induced upon terminal differentiation of murine primary calvarial osteoblasts. Using RT-PCR and Western Blotting we further demonstrated that differentiated osteoblasts, in contrast to neuronal cells, specifically express the short transmembrane isoform of Rptpzeta. To uncover a potential role of Rptpzeta in bone remodeling we next analyzed the skeletal phenotype of a Rptpzeta-deficient mouse model using non-decalcified histology and histomorphometry. Compared to wildtype littermates, the Rptpzeta-deficient mice display a decreased trabecular bone volume at the age of 50 weeks, caused by a reduced bone formation rate. Likewise, Rptpzeta-deficient calvarial osteoblasts analyzed ex vivo display decreased expression of osteoblast markers, indicating a cell-autonomous defect. This was confirmed by the finding that Rptpzeta-deficient osteoblasts had a diminished potential to form osteocyte-like cellular extensions on Matrigel-coated surfaces. Taken together, these data provide the first evidence for a physiological role of Rptpzeta in bone remodeling, and thus identify Rptpzeta as the first PTP regulating bone formation in vivo.

[Subdental synchondrosis. Computed tomographic and histologic investigation on morphological aspects of fracture at the base of the dens in 36 human axis specimens]. / Die subdentale Synchondrose. Eine computertomographische und histologische Untersuchung zu morphologischen Aspekten der Densbasisfraktur an 36 humanen Axispräparaten.

BACKGROUND: During development of the axis, four different ossification centers are formed. The two cranial ossification centers are demarcated from the ossification center of the vertebral corpus by a subdental synchondrosis. During further development the subdental synchondrosis--which is thought to close spontaneously--might not close completely, which leads to the necessity for differentiating synchondrotic remnants from a fracture at the base of the dens (type II according to Anderson and D'Alonzo). RESULTS: To characterize the architecture of the axis with particular attention to the subdental synchondrosis, the axis was harvested from 36 age- and gender-matched patients covering the human aging process from adolescence to senescence. In all specimens bone mineral density (BMD) was measured by peripheral quantitative computed tomography (pQCT). Morphological analysis after undecalcified processing of all specimens revealed a persistency of the subdental synchondrosis in 87% of all patients. Histological characterization of the subdental synchondrosis showed a cartilaginous structure interspersed with focal mineralization. Furthermore, static histomorphometric analysis revealed that trabecular bone volume and cortical thickness were significantly reduced within the base of the axis as compared to the dens and the corpus, respectively. CONCLUSION: Taken together, these results provide evidence that the base of the axis is a structurally distinct region. Besides well-recognized biomechanical aspects, these results suggest that the structure of the base of the axis might contribute to the occurrence of fractures of the axis and offer an additional explanation for the observation of nonunion after type II dens fractures.