Olaf Kilian

Bone ingrowth in bFGF-coated hydroxyapatite ceramic implants.

This experimental study was performed to evaluate angiogenesis, bone formation, and bone ingrowth in response to osteoinductive implants of bovine-derived hydroxyapatite (HA) ceramics either uncoated or coated with basic fibroblast growth factor (bFGF) in miniature pigs. A cylindrical bone defect was created in both femur condyles of 24 miniature pigs using a saline coated trephine. Sixteen of the 48 defects were filled with HA cylinders coated with 50 microg rhbFG, uncoated HA cylinders, and with autogenous transplants, respectively. Fluorochrome labelled histological analysis, histomorphometry, and scanning electron microscopy were performed to study angiogenesis, bone formation and bone ingrowth. Complete bone ingrowth into bFGF-coated HA implants and autografts was seen after 34 days compared to 80 days in the uncoated HA group. Active ring-shaped areas of fluorochrome labelled bone deposition with dynamic bone remodelling were found in all cylinders. New vessels could be found in all cylinders. Histomorphometric analysis showed no difference in bone ingrowth over time between autogenous transplants and bFGF-coated HA implants. The current experimental study revealed comparable results of bFGF-coated HA implants and autogenous grafts regarding angiogenesis, bone synthesis and bone ingrowth.

Expression of non-neuronal cholinergic system in osteoblast-like cells and its involvement in osteogenesis

Acetylcholine (ACh) is detected in a variety of non-neuronal cells where it acts as a para/autocrine signaling molecule controlling basic cell functions such as proliferation, differentation, and maintenance of cell-cell contacts. ACh-synthesizing enzymes include choline acetyltransferase and carnitine acetyltransferase (CarAT). ACh is released through vesicular exocytosis or directly from the cytoplasm via organic cation transporters (OCT). Extracellular ACh binds to nicotinic (nAChR) and muscarinic receptors (MR). Degradation of ACh is performed by acetylcholinesterase and butyrylcholinesterase (BChE). Here, we have determined whether these molecules are expressed in osteoblast-like cells, by means of reverse transcription polymerase chain reaction and immunohistochemistry, focusing on nAChR subunits α3 and α5. RNA for CarAT, OCT-1, M2R, M5R, nAChR subunits α3, α5, α9, α10, β2, β3, and BChE were detected in human (SAOS-2) and murine (MC3T3-E1) osteoblast-like cells. Other cholinergic components were only expressed species-specifically, e.g., M3R and nAChR subunit α7. Immunhistochemistry localized the nAChR subunits α3 and α5 in osteoblasts in vitro and in vivo where they were up-regulated after application of bone morphogenetic protein-2 (BMP-2) during fracture healing in a rat model. Thus, the cholinergic system of osteoblast-like cells might be regulated by BMP-2 during bone remodeling. Osteoblast-like cells express all necessary enzymes, transporters, and receptors for ACh synthesis and recycling.

Elution kinetics, antimicrobial efficacy, and degradation and microvasculature of a new gentamicin-loaded collagen fleece

Management of bone and soft tissue infections generally includes surgical procedures as well as attendant treatment and prevention with gentamicin-loaded fleeces. Conventional gentamicin-containing collagen fleeces currently in use are strongly acidic and exhibit limited biocompatibility thereby adversely affecting wound healing. To improve the antibiotic delivery system, a new phosphate-buffered, gentamicin-loaded fleece with pH-neutral properties has been developed (Jason G). This study aimed at comparing the elution kinetics of gentamicin release and the antimicrobial efficacy of conventional fleeces with the newly developed fleece in vitro. In addition, degradation and microvasculature of implanted fleeces were examined in a rat model and assessed using histology, as well as detection of ED-1 and PECAM-expression using immunohistochemistry. We show that the phosphate-buffered fleeces have reduced release (p < 0.05) of the integrated gentamicin. However, all of the fleeces tested had a significant antimicrobial effect on the growth of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa strains (p < 0.01). Among the fleeces tested, the new Jason G fleece had the weakest but nevertheless sufficient antimicrobial effectiveness. Evaluation of the antibiotic effect in the prevention of an infection showed no differences between the applied fleeces. Following surgical implantation of fleece in the backs of Wistar rats we observed, on day 5 after implantation, an increase in cell infiltration and microvascularization with the phosphate-buffered fleece as compared with conventional fleeces, which show necrotic cells on their surface. Unlike the acidic fleeces, on day 15 after implantation the pH-neutral fleece was resorbed widely. Here, we show that the new, pH-neutral, gentamicin-containing fleece Jason G exhibits good overall antimicrobial effectiveness against both gram-positive and gram-negative bacteria in vitro with improved degradation properties and microvasculature formation in vivo.

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 developped: 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.ZusammenfassungEinleitung: Nachdem Pilon-tibial-Frakturen über Jahrzehnte mit Plattenoteosynthesen versorgt wurden, ergaben sich in den letzten 10 Jahren differenzierte Behandlungsformen: Nach primärer interner Stabilisierung erfolgt heute (abhängig von Frakturtyp und Weichteilschaden) die Versorgung durch a) zweizeitiges Vorgehen (primärer Fixateur externe und sekundäre interne Fixation), b) alleinige Ausheilung im Fixateur externe, kombiniert mit Minimalosteosynthesen (Kirschner-Drähte, Lochschrauben), oder c) mit primärer Spongiosaplastik zur Vermeidung aseptischer Pseudarthrosen bei Stauchungsbrüchen. Methode: Von 151 Patienten, die von 1979 bis 1995 in unserer Klinik mit insgesamt 160 Pilon-tibial-Frakturen behandelt wurden, konnten 107 Patienten mit 113 Frakturen nachuntersucht werden. Verglichen wurden die Ergebnisse der C2-/C3-Frakturen, da nur hier alle 3 Verfahren zur Anwendung kamen. Ergebnisse:Über 75% waren geschlossene Frakturen (Weichteilschaden hauptsächlich G2 nach Oestern und Tscherne). Bei den offenen Frakturen fand sich am häufigsten Grad 3 nach Gustilo und Anderson. In 62 Fällen war der postoperative Verlauf komplikationsfrei; Frühkomplikationen waren im wesentlichen Weichteilinfektionen (25,7%), Spätkomplikationen vor allem Pseudarthrosen (25,7%). Am niedrigsten war die Infektionsrate bei alleiniger interner Fixierung, am höchsten beim zweizeitigen Vorgehen; allerdings wurden hier die besten klinischen Spätergebnisse erzielt. Schlußfolgerung: Die Komplikationsrate in der Behandlung der Pilon-tibial-Frakturen ist vom Frakturtyp,den begleitendem Weichteilschaden und dem Operationsverfahren abhängig. Bei geringem Weichteilschaden konnten mit der primären internen Osteosynthese gute Ergebnisse erreicht werden. Bei höhergradigem Weichteilschaden ließ sich die Weichteildeckung und die Gelenkflächenrekonstruktion mit einem zweizeitigen Verfahren gut lösen. Hierbei ist entscheidend, daß bei der Primäroperation (Fixateur externe) erheblich dislozierte Fragmente minimal-invasiv reponiert und mittels Kirschner-Drähten oder Lochschrauben fixiert werden. Die Fibulaosteosynthese ist zur Stabilisierung des zweiten Pfeilers der Sprunggelenkgabel bei der Primäroperation obligat.

Observations on the microvasculature of bone defects filled with biodegradable nanoparticulate hydroxyapatite

The microvascularization of metaphyseal bone defects filled with nanoparticulate, biodegradable hydroxyapatite biomaterial with and without platelet factors enrichment was investigated in a minipig model. Results from morphological analysis and PECAM-1 immunohistochemistry showed the formation of new blood vessels into the bone defects by sprouting and intussusception of pre-existing ones. However, no significant differences were observed in the microvascularization of the different biomaterials applied (pure versus platelet factors-enriched hydroxyapatite), concerning the number of vessels and their morphological structure at day 20 after operation. The appearance of VEGFR-2 positive endothelial progenitor cells in the connective tissue between hydroxyapatite particles was also found to be independent from platelet factors enrichment of the hydroxyapatite bone substitute. In both groups formation of lymphatic vessels was detected with a podoplanin antibody. No differences were noted between HA/PLF- and HA/PLF+ implants with respect to the podoplanin expression level, the staining pattern or number of lymphatic vessels. In conclusion, the present study demonstrates different mechanisms of blood and lymphatic vessel formation in hydroxyapatite implants in minipigs.

Die „frozen shoulder“Arthroskopische, histologische und elektronenmikroskopische Untersuchungen

Abstract.Introduction: During a period of 17 months (February 1998 to July 1999) arthroscopy was performed in 172 patients because of pathologic changes of the shoulder joint. 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 Dupuytrens 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.Zusammenfassung.Einleitung: Im Zeitraum von 17 Monaten (Februar 1998 bis Juli 1999) wurde bei 172 Patienten wegen krankhafter Veränderungen im Bereich des Schultergelenks eine Arthroskopie unter stationären Bedingungen durchgeführt. Methodik: Aufgrund der anamnestischen Erhebung und der klinischen Untersuchung erfüllten lediglich 11 Patienten die schon im Jahre 1969 von Lundberg [7] aufgestellten Kriterien einer primären „frozen shoulder“. Im Rahmen weiterführender diagnostischer Maßnahmen wie Röntgenuntersuchung, Sonographie und MRT ließen sich bei den 11 Patienten keine Veränderungen nachweisen, die eine Ursache für das klinische Bild der Schultersteife darstellen. Im Rahmen der Anamnese fand sich bei den 11 Patienten mit „frozen shoulder“ zusätzlich in 2 Fällen eine Hyperthyreose, in 5 Fällen ein Diabetes mellitus Typ II und in weiteren 5 Fällen eine Dupuytrensche Kontraktur. In Anlehnung an die in jüngsten Veröffentlichungen publizierte operative Vorgehensweise von Habermeyer [4], Ogilvie [10] und Warner [16] führten wir bei diesen Patienten eine arthroskopische Arthrolyse des kontrakten Schultergelenks durch. Ergebnisse: Im Rahmen der diagnostischen Arthroskopie ließen sich bei über 50 % der Patienten mit dem Krankheitsbild einer „frozen shoulder“ Synovialitiden im oberen und ventralen Gelenkbereich nachweisen. Intraarticuläre Adhäsionen fanden wir jedoch interessanterweise in keinem unserer Patienten, wobei der Recessus axillaris in keinem Fall obliteriert war. Insgesamt fand sich bei 7 der 11 Patienten mit „frozen shoulder“ eine auffällig kontrakte Bicepssehne, welche einen vermehrten Druck auf den darunterliegenden Humeruskopf ausübte, der in den korrespondierenden Arealen arthroskopisch sichtbare veränderte Knorpelbezirke aufwies. Eine deutliche Vermehrung fibroblastärer Zellen neben aufgelockerten Bindegewebearealen konnte im Rahmen der histologischen Untersuchung von Biopsaten aus der Kapsel im Rotatorenintervall dargestellt werden. Besonders auffällig waren verdickte Gefäßwände sowie eine Verbreiterung der synovialen Deckzellschicht, wobei in allen histologischen Proben keine Entzündungszeichen nachgewiesen werden konnten. Bei der transmissionselektronenmikroskopischen Untersuchung (TEM) der aus der Kapsel im Rotatorenintervall entnommenen Proben waren strukturelle Kollagenveränderungen zu erkennen, wobei typischerweise ein Verlust der Fibrillenordnung sowie eine Verdrillung der Kollagenfibrillen vorlagen. Diese Verdrillungen führten bis zu einem 4 fach vergrößerten Durchmesser der Kollagenfibrillen, wobei zwischen den aufgetriebenen Kollagenfibrillenbündeln wiederum dünne, elastinähnliche Filamente demonstriert wurden. Zusammenfassung: Im Stadium der primären „frozen shoulder“ konnten wir ausschließlich eine Fibrosierung der Gelenkkapsel nachweisen. Elektronenmikroskopisch waren Verdrillungen der Kollagenfibrillen auffällig.

New blood vessel formation and expression of VEGF receptors after implantation of platelet growth factor-enriched biodegradable nanocrystalline hydroxyapatite.

Vascular endothelial growth factor (VEGF) plays a key role for the interaction of osteoblasts and endothelial cells and, therefore, is an important factor for the osteointegration of bone substitutes. The aim of the current work was to study the effects of platelet growth factors (PLF) on new blood vessel formation and VEGF-receptors expression pattern in bone defects filled with nanocrystalline hydroxyapatite (HA) paste in miniature-pigs. Conventional histology, RT-PCR for VEGF and receptors mRNA, Western blot analysis, immunohistochemical staining and quantitative assessment of newly formed vessels was performed. HA enriched with platelet growth factor (HA/PLF+) led to an up-regulation of VEGF-R1 synthesis, a slightly enhanced number of newly formed vessels with higher sprouting activity compared with HA without PLF (HA/PLF−) filling defects. These observation are most likely attributable to a stimulating effect of TGF-β from the platelet factor on VEGF expression in osteoblasts.

Enhancement of bone formation in hydroxyapatite implants by rhBMP-2 coating

The combination of hydroxyapatite (HA) implants serving as osteoconductive scaffold with growth factors is an interesting approach for the improvement of bone defect healing. The purpose of this study was to test whether recombinant human bone morphogenetic protein-2 (rhBMP-2) coating of solid HA-implants improves bone formation in a cortical bone defect. Cylindrical trephine mill defects (diameter: 9.8 mm, depth: 10 mm) were created into the cortical tibia shaft of minipigs and subsequently filled either by plain HA cylinders (Endobon) or by rhBMP-2-coated HA cylinders. Fluorochrome labeling for the evaluation of time-dependent bone formation was done on days 8, 9, and 10 postsurgery with tetracyclin-100, at days 25 and 30 with alizarin-komplexon, and finally on days 32, 37, 73, and 79 with calcein green. Twelve weeks after implantation, the tibiae were harvested and were prepared for standard histological staining, fluorochrome analysis, and histomorphometry. Coating of HA implants with rhBMP-2 led to significant enhanced new bone formation of 84.7% (+/-4.6%) of the implant area with almost complete bony incorporation compared with only 27.7% (+/-8.5%) in the uncoated HA implants (p = 0.028). In both types of implants, osteoconduction of HA led to bone ingrowth of the surrounding host bone into the implants. However, only rhBMP-2-coated implants showed multitopic de novo bone formation reflecting the osteoinductive properties of rhBMP-2 in all areas of the HA implant. This study showed that the coating of HA ceramic implants with rhBMP-2 can significantly enhance new bone formation attributable to its osteoinductive effects.

Quantitative analyses of bone composition in acetylcholine receptor M3R and alpha7 knockout mice

AIMS Increasing collagen synthesis was observed in lung after stimulation of nicotinic and muscarinic acetylcholine receptors (nAChR and mAChR) on fibroblasts. Since collagen synthesis is an important process during fracture healing and bone remodelling, we asked whether cholinergic receptors are involved in bone collagen production. MAIN METHODS In the present study we analysed 16 week old male knockout mice for nAChRα7 (α7-KO) and mAChR M3R (M3R-KO) in correlation to their corresponding wild types (WT). Microarchitecture of right femora, vertebrae Th13 and L1 were analysed by 3D Micro-CT, left femora by a three-point bending test and humeri by real-time RT-PCR. KEY FINDINGS A significant decrease in relative bone volume, trabecular thickness, trabecular number, bone surface density, and a significant increase in trabecular separation and structure model index were measured for the M3R-KO using Micro-CT analysis. Bending stiffness of M3R-KO was significantly reduced in comparison to WT as well as the collagen 1α1 and 1α2 mRNA expression was down-regulated. No changes were detected for α7-KO using Micro-CT, biomechanical testing, and collagen mRNA expression. SIGNIFICANCE Our results indicate that nAChRα7 are not involved in the regulation of bone collagen synthesis whereas M3R exert stimulatory effects on cancellous bone microarchitecture, flexural rigidity, and bone matrix synthesis. Since the M3R-KO exhibit bone structures similar to systemically diseased bone it might be valuable to establish new therapeutic strategies using administration of agonists for the M3R to improve bone qualities.

Reconstruction of the lower leg with the sural artery flap.

Abstract Background. Soft tissue covering on the lower leg is frequently a difficult challenge and causes increasing problems for treatment in smaller hospitals. We introduce a plastic surgery method for covering these soft tissue defects. Method. An island of skin, centered above the sural nerve, is cut out in the middle dorsal area of the lower leg. The subcutaneous vascular pedicle is prepared along the nerve. The gained skin flap is rotated into the defect site and fixed without tension. The wound of the donor-site defect is usually closed primarily, and the flap pedicle is covered with Meshcraft. Results. In 18 of the 21 patients treated by this method the flap healed without functional impairment, one patient experienced a necrosis leading to the loss of the flap. And a partial loss of the skin island was recorded in two cases, complete healing was achieved by means of Meshcraft transplantation. Conclusions. The superficial sural artery flap usually results in a reliable and complete healing of soft tissue defects within the area of the lower legs with justifiable operational expenditures also for elderly patients and those with vascular ailments.