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The treatment of osteochondral defects in joint cartilage remains challenging due to its limited repair capacity. This study presents a metallic osteochondral plug with hydroxyapatite (HA)-coated cap edges for improved implant-tissue contact. The hypothesis was that improved attachment prevents from synovial fluid-influx and thereby avoids osteolysis and resulting implant instability. In total, 24 female, adult sheep were randomized into three groups. All animals received an Episealer®-implant in the medial condyle of the right knee. The implants were coated with two different HA versions or uncoated (control group). After 12 weeks, the implant-tissue connections were analysed radiologically and histologically. In general, the groups with the coated cap edges showed a better quality of tissue connection to the implant. The occurrence of gaps between tissue and implant was more seldom, the binding of calcified and hyaline cartilage to the cap was significantly better than in the uncoated group. A histomorphometrically measured lower amount of void space in these groups compared to the group with the uncoated edges confirmed that. The hypothesis of a tighter cartilage bone contact was confirmed. The HA coating of the implant’s cap edges resulted in better adherence of cartilage to the implant, which was not previously reported. In conclusion, this led to a better contact between implant and cartilage as well as neighbouring bone. In clinical routine, joint fluid is aggressive, penetrates through cartilage rifts, and promotes osteolysis and loosening of implants. The observed sealing effect will act to prevent joint fluid to get access to the implant–tissue interfaces. Joint fluid is aggressive, can cause osteolysis, and can, clinically cause pain. These effects are liable to decrease with these findings and will further the longevity of these osteochondral implants.

Impaired healing outcomes or even non-unions after bone injury are still a highly relevant problem in the daily clinical life. Especially within an aging population, the occurrence of bone fractures increases and thus novel treatment approaches to overcome compromised bone regeneration are needed. The gold standard to treat delayed or non-healing bone injuries is still the use of autologous bone grafts to foster regeneration. Besides its successful treatment outcome, it also has disadvantages: a second surgery is needed in order to harvest the bone material and the material is highly limited. Looking into the recent literature, a multitude of different research approaches were already conducted to identify new possible strategies to treat impaired bone regeneration: application of mesenchymal stromal cells, platelet lysates, growth factors, interference in the immune system, or bone formation stimulation by ultrasound. This review gives an overview of the treatment approaches actually performed in the clinic as well as at the bench in the context of compromised bone healing. It clearly highlights the complexity of the nature of non-healing bone fractures as well as patient-dependent factors influencing the healing process.

To allow bone defect regeneration, autologous bone grafting still represents the gold standard. However, autograft harvesting has limitations, including an additional surgery, donor site morbidity, and limited availability. Demineralized bone matrix (DBM) would represent an alternative, yet lacks sufficient osteoinductive properties. Combining DBM with a potent agent, such as bone morphogenetic protein-2 (BMP-2) might be a feasible alternative approach, optimizing an established grafting material with strong osteoinductive properties. A unique mixing device has been developed that enables perioperative handling to reach a homogeneous and standardized paste for bone defect filling. DBM proved in vitro to be a suitable carrier for BMP-2, with a documented release over 56 days at concentrations sufficient to stimulate osteogenic differentiation. At the end of the elution experiment, 56 days, bioactive BMP was still captured within the DBM. Using a sheep drill hole defect model, DBM perioperatively mixed with...

Large segmental bone defect reconstruction with sufficient functional restoration is one of the most demanding challenges in orthopaedic surgery. Available regenerative treatment options, as the vascularized bone graft transfer, the Masquelet technique or the Ilizarov distraction osteogenesis, are associated with specific indications and distinct limitations. As an alternative, a hollow cylindrical ceramic-polymer composite scaffold (β-tricalcium phosphate and poly-lactid co-ε- caprolactone), facilitating a strong surface guiding effect for tissue ingrowth (group 1; n = 6) was investigated here. In combination with an additional autologous, cancellous bone graft filling, the scaffold's ability to work as an open-porous membrane to improve the defect healing process was analysed (group 2; n = 6). A novel model of a critical size (40 mm) tibia osteotomy defect stabilized with an external hybrid-ring fixator, was established in sheep. Segmental defect regeneration and tissue organization in relation to the scaffold were analysed radiologically, (immune-) histologically, and with second-harmonic generation imaging 12 weeks after surgery. The scaffold's tubular shape and open-porous structure controlled the collagen fibre orientation within the bone defect and guided the following mineralization process along the scaffold surface. In combination with the osteoinductive stimulus, a unilateral bony bridging of the critically sized defect was achieved in one third of the animals. The external hybrid-ring fixator was appropriate for large segmental defect stabilization in sheep.

Bioactive coatings have the potential to improve the bony integration of mechanically loaded orthopedic ceramic implants. Using the concept of mimicking the natural bone surface, four different coatings of varying thickness on a zirconia toughened alumina (ZTA) ceramic implant were investigated regarding their osseointegration in a drill-hole model in sheep. The hypothesis that a bioactive coating of ZTA ceramics would facilitate cancellous bone integration was investigated. The bioactive coatings consisted of either a layer of covalently bound multi phosphonate molecules (chemical modification = CM), a nano hydoxyapatite coating (HA), or two different bioactive glass (BG) coatings in micrometer thickness, forming a hydroxyl-carbonate apatite layer on the implant surface in vivo (dip-coated 45S5 = DipBG; sol-gel 70S30C = SGBG). Coated surfaces were characterized by scanning electron microscopy and X-ray photoelectron spectroscopy. After 12 weeks, osseointegration was evaluated via mechanical push-out testing and histology. HA enhanced the maximum push-out force (HA: mean 3573.85 ± 1119.91 N; SGBG: mean 1691.57 ± 986.76 N; p = 0.046), adhesive shear strength (HA: mean 9.82 ± 2.89 MPA; SGBG: mean 4.57 ± 2.65 MPA; p = 0.025), and energy release rate (HA: mean 3821.95 ± 1474.13 J/mm2; SGBG: mean 1558.47 ± 923.47 J/mm2; p = 0.032) compared to SGBG. The implant-bone interfacial stiffness increased by CM compared to SGBG coating (CM: mean 6258.06 ± 603.80 N/mm; SGBG: mean 3565.57 ± 1705.31 n/mm; p = 0.038). Reduced mechanical osseointegration of SGBG coated implants could be explained histologically by a foreign body reaction surrounding the implants.

Scaffold architecture guides bone formation. However, in critical-sized long bone defects additional BMP-mediated osteogenic stimulation is needed to form clinically relevant volumes of new bone. The hierarchical structure of bone determines its mechanical properties. Yet, the micro- and nanostructure of BMP-mediated fast-forming bone has not been compared with slower regenerating bone without BMP. We investigated the combined effects of scaffold architecture (physical cue) and BMP stimulation (biological cue) on bone regeneration. It was hypothesized that a structured scaffold directs tissue organization through structural guidance and load transfer, while BMP stimulation accelerates bone formation without altering the microstructure at different length scales. BMP-loaded medical grade polycaprolactone-tricalcium phosphate scaffolds were implanted in 30mm tibial defects in sheep. BMP-mediated bone formation after 3 and 12 months was compared with slower bone formation with a scaffold alone after 12 months. A multiscale analysis based on microcomputed tomography, histology, polarized light microscopy, backscattered electron microscopy, small angle X-ray scattering and nanoindentation was used to characterize bone volume, collagen fiber orientation, mineral particle thickness and orientation, and local mechanical properties. Despite different observed kinetics in bone formation, similar structural properties on a microscopic and sub-micron level seem to emerge in both BMP-treated and scaffold only groups. The guiding effect of the scaffold architecture is illustrated through structural differences in bone across different regions. In the vicinity of the scaffold increased tissue organization is observed at 3 months. Loading along the long bone axis transferred through the scaffold defines bone micro- and nanostructure after 12 months.

In fracture healing, skeletal and immune system are closely interacting through common cell precursors and molecular mediators. It is thought that the initial inflammatory reaction, which involves migration of macrophages into the fracture area, has a major impact on the long term outcome of bone repair. Interestingly, macrophages reside during all stages of fracture healing. Thus, we hypothesized a critical role for macrophages in the subsequent phases of bone regeneration. This study examined the impact of in vivo induced macrophage reduction, using clodronate liposomes, on the different healing phases of bone repair in a murine model of a standard closed femoral fracture. A reduction in macrophages had no obvious effect on the early fracture healing phase, but resulted in a delayed hard callus formation, thus severely altering endochondral ossification. Clodronate treated animals clearly showed delayed bony consolidation of cartilage and enhanced periosteal bone formation. Therefore, we decided to backtrack macrophage distribution during fracture healing in non-treated mice, focusing on the identification of the M1 and M2 subsets. We observed that M2 macrophages were clearly prevalent during the ossification phase. Therefore enhancement of M2 phenotype in macrophages was investigated as a way to further bone healing. Induction of M2 macrophages through interleukin 4 and 13 significantly enhanced bone formation during the 3 week investigation period. These cumulative data illustrate their so far unreported highly important role in endochondral ossification and the necessity of a fine balance in M1/M2 macrophage function, which appears mandatory to fracture healing and successful regeneration.

The influence of basal graft support combined to early loading following an osteochondral autograft procedure is unclear. It was hypothesized that bottomed grafts may allow for early mobilization by preventing graft subsidence and leading to better healing. Osteochondral autografts were press fitted in the femoral condyles of 24 sheep (one graft per animal). In the unbottomed group (n = 12), a gap of 2 mm was created between graft and recipient bone base. In the bottomed group (n = 12), the graft firmly rested on recipient bone. Animals were allowed immediate postoperative weightbearing. Healing times were 3 and 6 months per group (n = 6 per subgroup). After killing, histological and histomorphometric analyses were performed. Unbottomed grafts at 3 months showed significantly more graft subsidence (P = 0.024), significantly less mineralized bone (P = 0.028) and significantly worse cartilage and subchondral bone plate healing (P = 0.034) when compared to bottomed grafts. At 6 months, no differences were seen. Compared to the native situation, unbottomed grafts showed significantly more graft subsidence (P = 0.024), whereas bottomed grafts did not. Cystic lesions were seen in both groups. Osteoclasts were closely related to the degree of bone remodelling. In the animal model, in the case of early loading, bottomed osteochondral autografts have less chance of graft subsidence. Evident subsidence negatively influences the histological healing process. In the osteochondral autograft procedure, full graft support should be aimed for. This may allow for early mobilization, diminish graft subsidence and improve long-term integration.

The transplantation of autologous bone graft as a treatment for large bone defects has the limitation of harvesting co-morbidity and limited availability. This drives the orthopaedic research community to develop bone graft substitutes. Routinely, supra-physiological doses of bone morphogenetic proteins (BMPs) are applied perpetuating concerns over undesired side effects and cost of BMPs. We therefore aimed to design a composite scaffold that allows maintenance of protein bioactivity and enhances growth factor retention at the implantation site. Critical-sized defects in sheep tibiae were treated with the autograft and with two dosages of rhBMP-7, 3.5 mg and 1.75 mg, embedded in a slowly degradable medical grade poly(ε-caprolactone) (PCL) scaffold with β-tricalcium phosphate microparticles (mPCL-TCP). Specimens were characterised by biomechanical testing, microcomputed tomography and histology. Bridging was observed within 3 months for the autograft and both rhBMP-7 treatments. No significant difference was observed between the low and high rhBMP-7 dosages or between any of the rhBMP-7 groups and autograft implantation. Scaffolds alone did not induce comparable levels of bone formation compared to the autograft and rhBMP-7 groups. In summary, the mPCL-TCP scaffold with the lower rhBMP-7 dose led to equivalent results to autograft transplantation or the high BMP dosage. Our data suggest a promising clinical future for BMP application in scaffold-based bone tissue engineering, lowering and optimising the amount of required BMP.

Experimental data indicate that hypercapnic adidosis has anti-inflammatory effects. These anti-inflammatory effects may even be a beneficial property in case of low tidal volume ventilation with consecutive hypercapnic acidosis. It is unclear whether these anti-inflammatory effects predominate in critically ill patients who suffer from multiple pro- and anti-inflammatory insults like extracorporeal organ support (pro-inflammatory), metabolic acidosis (pro- and anti-inflammatory), as well as hypoxia (pro-inflammatory). Eighteen pigs were randomized into three groups, mechanically ventilated and connected to a continuous veno-venous hemofiltration (CVVH) as pro-inflammatory insult. A reference group with normal acid-base state obtained normoventilation; a normoxemic acidemia group obtained normoxemic, mixed acidemia due to infusion of lactic and hyperchloremic acid and low tidal volume ventilation, and in a hypoxemic acidemia group the mixed acidemia was paralleled by hypoxemia. Lung histology including pulmonary leukocyte invasion, blood gases, blood cell counts, and hemodynamics were examined. The histological examination of the lungs of acidemic pigs showed a suppressed invasion of leukocytes and thinner alveolar walls compared with normoventilated and with hypoxemic pigs. Enhanced congestion and alveolar red blood cells (RBCs) combined with an increase of the pulmonary artery pressure were observed in acidemic pigs in comparison with the reference group. Normoxemic acidemia reduced the pro-inflammatory reaction to the CVVH and mechanical ventilation in the ventilated lung areas in the form of pulmonary leukocyte invasion. However, this did not result in reduced scores for lung injury. Instead, an increased score for criteria which represent lung injury (congestion and alveolar RBCs) was observed in acidemic pigs.

Fracture treatment is an old endeavour intended to promote bone healing and to also enable early loading and regain of function in the injured limb. However, in today’s clinical routine the healing potential of the initial fracture haematoma is still not fully recognized. The Arbeitsgemeinschaft für Osteosynthesefragen (AO) formed in Switzerland in 1956 formulated four AO principles of fracture treatment which are still valid today. Fracture treatment strategies have continued to evolve further, as for example the relatively new concept of minimally invasive plate osteosynthesis (MIPO). This MIPO treatment strategy harbours the benefit of an undisturbed original fracture haematoma that supports the healing process. The extent of the supportive effect of this haematoma for the bone healing process has not been considered in clinical practice so far. The rising importance of osteoimmunological aspects in bone healing supports the essential role of the initial haematoma as a source for inflammatory cells that release the cytokine pattern that directs cell recruitment towards the injured tissue. In reviewing the potential benefits of the fracture haematoma, the early development of angiogenic and osteogenic potentials within the haematoma are striking. Removing the haematoma during surgery could negatively influence the fracture healing process. In an ovine open tibial fracture model the haematoma was removed 4 or 7 days after injury and the bone that formed during the first two weeks of healing was significantly reduced in comparison with an undisturbed control. These findings indicate that whenever possible the original haematoma formed upon injury should be conserved during clinical fracture treatment to benefit from the inherent healing potential.

Bone development is influenced by the local mechanical environment. Experimental evidence suggests that altered loading can change cell proliferation and differentiation in chondro- and osteogenesis during endochondral ossification. This study investigated the effects of three-point bending of murine fetal metatarsal bone anlagen in vitro on cartilage differentiation, matrix mineralization and bone collar formation. This is of special interest because endochondral ossification is also an important process in bone healing and regeneration. Metatarsal preparations of 15 mouse fetuses stage 17.5 dpc were dissected en bloc and cultured for 7 days. After 3 days in culture to allow adherence they were stimulated 4 days for 20 min twice daily by a controlled bending of approximately 1000-1500 microstrain at 1 Hz. The paraffin-embedded bone sections were analyzed using histological and histomorphometrical techniques. The stimulated group showed an elongated periosteal bone collar while the total bone length was not different from controls. The region of interest (ROI), comprising the two hypertrophic zones and the intermediate calcifying diaphyseal zone, was greater in the stimulated group. The mineralized fraction of the ROI was smaller in the stimulated group, while the absolute amount of mineralized area was not different. These results demonstrate that a new device developed to apply three-point bending to a mouse metatarsal bone culture model caused an elongation of the periosteal bone collar, but did not lead to a modification in cartilage differentiation and matrix mineralization. The results corroborate the influence of biophysical stimulation during endochondral bone development in vitro. Further experiments with an altered loading regime may lead to more pronounced effects on the process of endochondral ossification and may provide further insights into the underlying mechanisms of mechanoregulation which also play a role in bone regeneration.

Local mechanical conditions are known to play a role during the regeneration of musculoskeletal tissues, and histomorphometrical investigations of the time course of healing have enabled specific conclusions regarding the mechanosensitivity of tissue differentiation. However, the mechanism for this influence is not clearly understood. In order to extend this analysis, it is essential to link local histological understanding with direct characterisation of the local mechanical environment. Digital image correlation (DIC) is a computer-based image analysis technique that enables the non-contact measurement of strains on material surfaces and is finding application in many areas of biomechanics. Here we report a DIC technique to investigate the local distribution of mechanical strain within regenerating soft tissue sections. We provide exemplary data from analysis of a section of sheep bone callus. An assessment of displacement measurement accuracy gave an RMS error of 4.2 microm, corresponding to an estimated strain error of 1.4%. The sections showed concentrations of up to four times the applied strain and comparison of the strain patterns with histological analysis confirmed that these concentrations reflected boundaries between hard and soft callus.

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disease resulting from inactivating mutations in the gene encoding the protein neurofibromin. NF1 manifests as a heritable susceptibility to tumours of neural tissue mainly located in the skin (neurofibromas) and pigmented skin lesions. Besides these more common clinical manifestations, many NF1 patients (50%) have abnormalities of the skeleton. Long bones are often affected (usually the tibia) and the clinical signs range from bowing to spontaneous fractures and non-unions. Here we present the analysis of bone fracture healing in the Nf1(Prx1)-knock-out mouse, a model of NF1 long bone dysplasia. In line with previously reported cortical bone injury results, fracture healing was impaired in Nf1(Prx1) mice. We showed that the defective fracture healing in Nf1(Prx1) mice is characterized by diminished cartilaginous callus formation and a thickening of the periosteal bone. These changes are paralleled by fibrous tissue accumulation within the fracture site. We identify a population of fibrous tissue cells within the Nf1 deficient fracture as alpha-smooth muscle actin positive myofibroblasts. Additionally, histological and in-situ hybridization analysis reveal a direct contact of the fracture site with muscle fascia, suggesting a possible involvement of muscle derived cells in the fracture deterioration.

Nowadays total joint replacement is an indispensable component of modern medicine. The surfaces characteristics of cementless prostheses may be altered to achieve an accelerated and enduring bony integration. Classic surface coatings bear the risk of loosening or flaking from the implant body. This risk is excluded by the chemical conversion of the naturally existing TiO2 surface layer into calcium titanate. The aim of this experimental animal study was to investigate the bony integration of implants with a new calcium titanate surface (Ca4Ti3O10) compared with a conventional standard Ti6Al4V surface. Cylindrical implants, made of titanium alloy (Ti6Al4V) were implanted in both lateral femoral condyles of New Zealand white rabbits. In each animal, an implant with and without surface treatment was inserted in a blinded manner. Animals were sacrificed after 4, 12, and 36 weeks, respectively. The axial pull-off forces were determined for 25 animals using a universal testing machine (Zwick Z010, Ulm, Germany). Furthermore, a histological analysis of the bony integration of the implants was performed in 12 specimens. In general, the pull-off forces for untreated and treated implants increased with longer survival times of the rabbits. No significant difference could be shown after 4 weeks between treated and untreated implants. After 12 weeks, the treated implants revealed a statistical significant higher pull-off force. After 36 weeks, the pull-off forces for treated and untreated implants aligned again. Titanium implants treated with calcium titanate, may offer an interesting and promising implant surface modification for endoprosthetic implants. They might lead to an accelerated osseointegration of total hip and knee replacements. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 100A:2710–2716, 2012.

During hematoma formation following injury, an inflammatory reaction ensues as an initial step in the healing process. As granulation tissue matures, revascularization is a prerequisite for successful healing. The hypothesis of this study was that scarless tissue reconstitution in the regenerative bone healing process is dependent on a balanced immune reaction that initiates revasculatory steps. To test this hypothesis, cellular composition and expression profiles of a bone hematoma (regenerative, scarless) was compared with a muscle soft tissue hematoma (healing with a scar) in a sheep model. Upregulation of regulatory T helper cells and anti-inflammatory cytokine expression (IL-10) coincided with an upregulation of angiogenic factors (HIF1α and HIF1α regulated genes) in the regenerative bone hematoma but not in the soft tissue hematoma. These results indicate that the timely termination of inflammation and early onset of revascularization are interdependent and essential for a regenerative healing process. Prolonged pro-inflammatory signaling occurring in a delayed bone-healing model supports the finding that timely termination of inflammation furthers the regenerative process. Differing cellular compositions are due to different cell sources invading the hematoma, determining the ensuing cytokine expression profile and thus paving the path for regenerative healing in bone or the formation of scar tissue in muscle injury.

ZusammenfassungDie Knochenheilung wird durch einen komplexen, kaskadenartig ablaufenden Regenerationsmechanismus ermöglicht, der nicht mit der Bildung einer Narbe, sondern mit der vollständigen Wiederherstellung der ursprünglichen Struktur und Funktion des Knochens endet. Die Untersuchung dieses komplexen Prozesses ist nur im Tiermodell umfassend möglich, weshalb es diverse Modelle in Großund Kleintier zur Frakturheilung gibt. Eine histologische Analyse des Frakturgebiets kann den Ablauf der Knochenheilung sehr exakt und standardisiert beschreiben. Hier sollen der physiologische Ablauf der Frakturheilung in zwei gebräuchlichen Modellen (Schaf und Ratte) histologisch beschrieben und die Stärken einer histologischen Analyse herausgehoben werden.

ZusammenfassungUm Frakturheilungszeiten zu minimieren und Patienten eine möglichst schnelle und weitestgehende Rehabilitation zu ermöglichen, ist ein Verständnis sowohl der biologischen Prozesse der Heilung als auch der mechanischen Bedingungen, unter denen diese Heilung vonstatten geht, nötig. Wissen um die mechanischen Bedingungen ist insbesondere deshalb von Bedeutung, da die biologischen Prozesse der Heilung und Regeneration von den durch Muskelund Gelenkkräfte definierten Rahmenbedingungen wesentlich beeinflusst werden. Durch die Wahl der Osteosynthese können die mechanischen Rahmenbedingungen an der Fraktur und dem physikalischen Stimulus, dem die zellulären Strukturen ausgesetzt sind, weiter moduliert werden. Unabhängig von der Methode der Fraktur-stabilisierung ist die sekundäre Knochenheilung immer einer komplexen interfragmentären Bewegung ausgesetzt. Dabei beeinflussen die auftretenden Fragmentbewegungen den Heilungsprozess in seiner Art und Schnelligkeit.

Bone healing is known to occur through the successive formation and resorption of various tissues with different structural and mechanical properties. To get a better insight into this sequence of events, we used environmental scanning electron microscopy (ESEM) together with scanning small-angle X-ray scattering (sSAXS) to reveal the size and orientation of bone mineral particles within the regenerating callus tissues at different healing stages (2, 3, 6, and 9 weeks). Sections of 200 µm were cut from embedded blocks of midshaft tibial samples in a sheep osteotomy model with an external fixator. Regions of interest on the medial side of the proximal fragment were chosen to be the periosteal callus, middle callus, intercortical callus, and cortex. Mean thickness (T parameter), degree of alignment (ρ parameter), and predominant orientation (ψ parameter) of mineral particles were deduced from resulting sSAXS patterns with a spatial resolution of 200 µm. 2D maps of T and ρ overlapping with ESEM images revealed that the callus formation occurred in two waves of bone formation, whereby a highly disordered mineralized tissue was deposited first, followed by a bony tissue with more lamellar appearance in the ESEM and where the mineral particles were more aligned, as revealed by sSAXS. As a consequence, degree of alignment and mineral particle size within the callus increased with healing time, whereas at any given moment there were structural gradients, for example, from periosteal toward the middle callus.

Research regarding the potency and potential of the fracture hematoma has begun to receive increasing attention. However, currently there is a paucity of relevant literature on the capability and composition of the fracture hematoma. This review briefly summarizes the regenerative fracture healing process and the close interplay between the skeletal and immune systems. The role of immune cells in wound healing is also discussed to clarify their involvement in immunological processes during regeneration. We attempt to describe the current state of knowledge regarding the fracture hematoma as the initial stage of the regenerative process of fracture healing. The review discusses how a better understanding of immune reactions in the hematoma may have implications for bone tissue engineering strategies. We conclude the review by emphasizing how additional investigations of the initial phase of healing will allow us to better differentiate between deleterious and beneficial aspects of inflammation, thereby facilitating improved fracture treatment strategies.

The objectives of this study were to (1) establish a reproducible atrophic non-union model in rats by creation of a segmental femoral bone defect that allows, (2) in-depth characterization of impaired healing, and (3) contrast its healing patterns to the normal course. Hypothesis was that a 5-mm bone defect in male rats would deviate from uneventful healing patterns and result in an atrophic non-union. A femoral osteotomy was performed in two groups of 12-week-old male rats (1 vs. 5 mm gap) stabilized with an external fixator. Bone healing in these models was evaluated by radiology, biomechanics, and histology at 6 or 8 weeks. The evaluation of the 5-mm group revealed in some cases a delayed rather than a non-union, and therefore, a group of female counterparts was included. The creation of a 5-mm defect in female rats resulted in a reproducible atrophic non-union characterized by sealing of the medullary canal, lack of cartilage formation, and negligible mechanical properties of the callus. In both gap size models, the male subjects showed advanced healing compared to females. This study showed that even under uneventful healing conditions in terms of age and bone defect size, there is a sex-specific advanced healing in male compared to female subjects. Contrary to our initial hypothesis, only the creation of a 5-mm segmental femoral defect in female rats led to a reproducible atrophic non-union. It has been shown that an atrophic non-union exhibits different healing patterns compared to uneventful healing. A total lack of endochondral bone formation, soft tissue prolapse into the defect, and bony closure of the medullary cavity have been shown to occur in the non-union model.

Secondary fracture healing in long bones leads to the successive formation of intricate patterns of tissues in the newly formed callus. The main aim of this work was to quantitatively describe the topology of these tissue patterns at different stages of the healing process and to generate averaged images of tissue distribution. This averaging procedure was based on stained histological sections (2, 3, 6, and 9 weeks post-operatively) of 64 sheep with a 3 mm tibial mid-shaft osteotomy, stabilized either with a rigid or a semi-rigid external fixator. Before averaging, histological images were sorted for topology according to six identified tissue patterns. The averaged images were obtained for both fixation types and the lateral and medial side separately. For each case, the result of the averaging procedure was a collection of six images characterizing quantitatively the progression of the healing process. In addition, quantified descriptions of the newly formed cartilage and the bone area fractions (BA/TA) of the bony callus are presented. For all cases, a linear increase in the BA/TA of the bony callus was observed. The slope was greatest in the case of the most rigid stabilization and lowest in the case of the least stiff. This topological description of the progression of bone healing will allow quantitative validation (or falsification) of current mechano-biological theories.

ZusammenfassungDer Schrauben-Knochen-Kontakt ist die Schwachstelle des externen Fixateurs, der die Funktionalität der Osteosynthese und den Behandlungserfolg bedingt. Daher ist eine stabile Integration der Schrauben im Knochen sowohl initial als auch über den Behandlungsverlauf essenziell. Die Hypothese der Studie war, dass das Auftreten von Schraubenlockerungen und -infekten mit der Implantationsdauer ansteigt, während die generelle Stabilität der Schrauben-Knochen-Verbindung mit der Zeit abnimmt. Für die Studie wurde bei 24 Schafen eine Tibia-Osteotomie vorgenommen und mit einem externen Fixateur stabilisiert. Die Schrauben-Knochen-Verbindung wurde nach drei, sechs und neun Wochen Standzeit radiologisch, biomechanisch, mikrobiologisch und histologisch untersucht. Entgegen der Hypothese blieb die Stabilität der Schrauben-Knochen-Verbindung während der Standzeit un-verändert. Dieser unerwartete Effekt könnte auf ein zunehmendes Remodeling im Knochen um die Schrauben zurückzuführen sein, begünstigt durch eine intensive Wundpflege und eine damit einhergehende niedrige Infektrate.

Blood vessel formation is a prerequisite for bone healing. In this study, we tested the hypothesis that a delay in bone healing is associated with an altered regulation of blood vessel formation. A tibial osteotomy was performed in two groups of sheep and stabilized with either a rigid external fixator leading to standard healing or with a highly rotationally unstable one leading to delayed healing. At days 4, 7, 9, 11, 14, 21, and 42 after surgery, total RNA was extracted from the callus. Gene expressions of vWF, an endothelial cell marker, and of several molecules related to blood vessel formation were studied by qPCR. Furthermore, histology was performed on fracture hematoma and callus sections. Histologically, the first blood vessels were detected at day 7 in both groups. mRNA expression levels of vWF, Ang1, Ang2, VEGF, CYR61, FGF2, MMP2, and TIMP1 were distinctly lower in the delayed compared to the standard healing group at several time points. Based on differential expression patterns, days 7 and 21 postoperatively were revealed to be essential time points for vascularization of the ovine fracture callus. This work demonstrates for the first time a differential regulation of blood vessel formation between standard and mechanically induced delayed healing in a sheep osteotomy model.

Bone fracture leads to a cycle of inflammation, cellular migration, and proliferation to restore tissue integrity. Immune cells at the site of injury are involved especially in the early phase of the healing process, but little is known about the cells present in the initial fracture hematoma. The hypothesis of this study was that the cellular composition in a fracture hematoma differs from that found in a muscle hematoma and that these divergences get more pronounced over time. By using a reproducible osteotomy model and muscle trauma in sheep the distributions of the immune cell subpopulations were evaluated 1 and 4 h after surgery. The cell amount within the first 4 h increased in both hematoma. The number of dead cells was higher in the muscle hematoma. One hour postoperatively the initial fracture hematoma revealed a lower granulocyte percentage compared to the muscle hematoma. The ratio of T helper to cytotoxic T cells was higher in the fracture hematoma compared to the muscle hematoma at both investigated time points. B cell percentage increased in the fracture but not in the muscle hematoma from 1 to 4 h. This is the first study that compares the immune cell subpopulations of a fracture and muscle hematoma.

To analyze the spectrum of findings in indirect MR-arthrography following osteochondral autograft transfer system (OATS) and crushed bone graft using a magnetic resonance imaging (MRI) scoring and grading system in relation to histology as the standard of reference.Iatrogenic lesions were set at ovine condylar facets (n=6/group), treated with OATS or crushed bone graft. 1.5 T MRI was performed 6 months after surgery using PD-weighted (w fat saturated (fs) fast spin echo (FSE), T1-w 2D, and 3D fs gradient echo (GE) sequences 30 min. after i.v. Gd-DTPA administration and passive joint exercise. The repair tissue was evaluated by two independent radiologists. The MR findings were compared to histology.In all cases, MRI and histologic grading correlated well and showed significant superior repair in OATS at 6 months (p0.05), reproducing the original articular contour and a good subchondral restoration. FsT1-w3DGE proved most appropriate identifying characteristic post-operative findings: the OATS group demonstrated bone marrow edema at the donor site and the graft/host interface showed significant enhancement in indirect MR-arthrography, indicating fibrocartilage. After crushed bone graft, we found an irregular structure and significant contrast uptake, consistent with remnants of bone grafts surrounded by inflammatory tissue.Indirect MR-arthrography is an accurate, non-invasive monitoring tool following OATS and crushed bone graft as the MRI scoring and grading system allows a reliable evaluation of normal and pathological osteochondral repair with a high histologic correlation.

BackgroundIn osteochondral defects, subchondral bone, as a load-bearing structure, is believed to be important for bone and cartilage regeneration.HypothesisA stiff scaffold creates better conditions for bone formation and cartilage regeneration than does a softer one.Study DesignControlled laboratory study.MethodsCritical osteochondral defects were created in the femoral condyles of 24 sheep. Subchondral bone was reconstructed with a stiff scaffold or a modified softer one, with untreated defects serving as controls. The repair response was evaluated with mechanical, histological, and histomorphometrical techniques at 3 and 6 months postoperatively.ResultsThe elastic modulus of regenerated fibrocartilage over the stiff scaffold tended to be higher than in the soft scaffold group (61 % vs 46% of healthy cartilage) at 3 months. No difference was determined at 6 months; all were well below healthy cartilage. Treated defects showed substantial degradation of the soft scaffold with surrounding sclerotic bone at 3 and 6 months. In contrast, degradation of the stiff scaffold was slower and occurred together with continuous osseous replacement.ConclusionStiff scaffolds were found to improve bone regeneration. In contrast, soft scaffolds provided less support, and consequently subchondral bone became sclerotic. Although regenerated cartilage formed over the stiff scaffolds at 3 months, and these exhibited better mechanical properties than did the soft scaffold group, the mechanical properties in both treated groups were the same at 6 months, not dissimilar to that of tissue formed in the untreated specimens and inferior to native articular cartilage.Clinical RelevanceThe results imply that subchondral defect filling in clinical settings advances bone regeneration and should have a comparable stiffness to that of healthy subchondral bone rather than being too flexible. Degradation of resorbable materials and consequently the loss of stiffness may compromise the healing of critical defects.

Adequate blood supply and sufficient mechanical stability are necessary for timely fracture healing. Damage to vessels impairs blood supply; hindering the transport of oxygen which is an essential metabolite for cells involved in repair. The degree of mechanical stability determines the mechanical conditions in the healing tissues. The mechanical conditions can influence tissue differentiation and may also inhibit revascularization. Knowledge of the actual conditions in a healing fracture in vivo is extremely limited. This study aimed to quantify the pressure, oxygen tension and temperature in the external callus during the early phase of bone healing. Six Merino-mix sheep underwent a tibial osteotomy. The tibia was stabilized with a standard mono-lateral external fixator. A multi-parameter catheter was placed adjacent to the osteotomy gap on the medial aspect of the tibia. Measurements of oxygen tension and temperature were performed for ten days post-op. Measurements of pressure were performed during gait on days three and seven. The ground reaction force and the interfragmentary movements were measured simultaneously. The maximum pressure during gait increased (p=0.028) from three (41.3 [29.2-44.1] mm Hg) to seven days (71.8 [61.8-84.8] mm Hg). During the same interval, there was no change (p=0.92) in the peak ground reaction force or in the interfragmentary movement (compression: p=0.59 and axial rotation: p=0.11). Oxygen tension in the haematoma (74.1 mm Hg [68.6-78.5]) was initially high post-op and decreased steadily over the first five days. The temperature increased over the first four days before reaching a plateau at approximately 38.5 degrees C on day four. This study is the first to report pressure, oxygen tension and temperature in the early callus tissues. The magnitude of pressure increased even though weight bearing and IFM remained unchanged. Oxygen tensions were initially high in the haematoma and fell gradually with a low oxygen environment first established after four to five days. This study illustrates that in bone healing the local environment for cells may not be considered constant with regard to oxygen tension, pressure and temperature.

The objective of this study was to examine the effects of low-intensity pulsed ultrasound treatment of murine fetal metatarsal (MT) bone anlagen in vitro. Metatarsal preparations of 17 mice fetuses stage 17.5 dpc were dissected en bloc and cultured for 7 days with and without low-intensity ultrasound stimulation. The total length of the metatarsal rudiments and the length of the calcified diaphysis were measured at days 1, 3, 5, and 7. After 7 days in culture, histological and histomorphometric analyses were performed. The increase in total length of the metatarsal bones and in length of the calcified diaphysis during in vitro culture was not affected by ultrasound treatment. Histological analysis of the MT preparations after 7 days of in vitro culture showed a healthy appearance of all specimens and revealed no differences in the general histological outcome between the stimulated and control groups. All histomorphometric parameters were unaffected by ultrasound stimulation, except for the length of the proximal hypertrophic zone which was significantly shorter in the stimulated bones compared to controls (p=0.043). Our results illustrate no stimulating effect of ultrasound treatment on endochondral ossification which may be based on different experimental conditions in comparison to other studies demonstrating a positive effect of sonication. Thus, ultrasonically induced stimulatory effects on endochondral ossification seem to be highly dependent on experimental conditions.

Background New tissue engineering strategies for bone regeneration need to be investigated in a relevant preclinical large animal model before making the translation into human patients. Therefore, our interdisciplinary group established a simplified large animal screening model for intramembranous bone defect regeneration in cancellous and cortical bone. Methods Related to a well-established model of cancellous drill hole defect regeneration in sheep, both the proximal and distal epimetaphyseal regions of the femur and the humerus were used bilaterally for eight drill hole cancellous defects (Ø 6 mm, 15 mm depth). Several improvements of the surgical procedure and equipment for an easier harvest of samples were invented. For the inclusion of cortical defect regeneration, a total of eight unicortical diaphyseal drill holes (6 mm Ø) were placed in the proximal-lateral and distal-medial parts of the metacarpal (MC) and metatarsal (MT) diaphyseal bone bilaterally. Acting moments within a normal gait cycle in the musculoskeletal lower limb model were compared with the results of the biomechanical in vitro torsion test until failure to ensure a low accidental fracture risk of utilized bones (ANOVA, p

Background: Bone-healing is known to be sensitive to the mechanical stability of fixation. However, the influence on healing of the individual components of fixation stiffness remains unclear. The aim of this study was to investigate the relationship between the initial in vitro fixation stiffness and the strength and stiffness of the callus after nine weeks. We hypothesized that axial stiffness would determine the healing outcome. Methods: A standardized midshaft osteotomy of the right tibia was performed on Merino-mix sheep and was stabilized with either one of four monolateral external fixators or one of two tibial nails inserted without reaming. The in vitro stiffness of fixation was determined in six loading conditions (axial compression, torsion, as well as bending and shear in the anteroposterior and mediolateral planes) on ovine tibial specimens. Stiffness was calculated by relating displacements of the fracture fragments, determined by means of attached optical markers, and the loads applied by a materials testing machine. Torsional testing until failure of the explanted tibiae was performed with use of a standard materials testing machine after nine weeks of healing to determine the failure moment and the torsional stiffness of the healed tibia. Results: External fixation in sheep generally resulted in higher fixation stiffness than did conventional unreamed tibial nailing. The use of angle-stable locking screws in tibial nailing resulted in fixation stiffness comparable with that of external fixation. The highest torsional moment to failure was observed for the external fixator with moderate axial stiffness and high shear stiffness. The fixator with the highest axial stability did not result in the highest failure moment. Low axial stability in combination with low shear stability resulted in the lowest failure moment. Conclusions: In this study, a clear relationship between the stability of fixation and the mechanical strength of the healing tibia was seen. Moderate levels of axial stability were associated with the highest callus strength and stiffness. Clinical Relevance: Optimizing axial stability and limiting shear instability appear to be important for creating conditions for timely fracture-healing.

Hyaline joint cartilage has only a limited potential for self-repair. Some of the published techniques for osteochondral defect therapy try to improve that potential. In this study, it was hypothesised that one of those surgical techniques, the crushed transplanted bone graft together with a collagen membrane, accelerates significantly the reconstruction of the subchondral bone plate and improves the mechanical and histological quality of repaired cartilage in osteochondral defects compared to an empty control defect. In order to test this hypothesis, defects were created in the left knee of 12 sheep and filled either with autologous crushed bone graft or left empty. The animals were sacrificed after 3 (n = 6) and 6 (n = 6) months. No differences were found either macroscopically or histomorphometrically between the bone graft and empty control defects. The biomechanical as well as the histological results of the bone graft defects were inferior to the control defects with inflammatory processes caused either by bone graft or membrane remnants. Based on the results in this sheep model, the filling of subchondral bone defects with compacted cancellous bone should be carefully reconsidered.

Although the sheep has become a standard model for understanding the mechanical conditions that occur after injury and investigating surgical treatments such as osteochondral defect healing and ligament reconstruction, no study has yet evaluated the contact forces that occur in the sheep tibio-femoral joint in vivo. In this study, bone pins, together with reflective markers, were used to measure the 3D kinematics of three sheep hind limbs, simultaneously with the ground reaction forces during repetitions of gait trials. Joint contact forces were then calculated using inverse dynamics and optimisation techniques. Whilst average peak axial tibio-femoral contact forces of 2.1 body weight (BW) were calculated across the 3 sheep, only small medio-lateral and antero-posterior shear forces, averaging 0.7 BW, were determined. Average knee flexion angles ranging from 49 degrees to 70 degrees were observed. From the forces determined in this study, we have provided a better understanding of the mechanical loading environment that occurs in sheep. This has important implications for the interpretation of knee studies in quadrupeds and their relevance to the clinical situation.

The formation of new blood vessels is a prerequisite for bone healing. CYR61 (CCN1), an extracellular matrix-associated signaling protein, is a potent stimulator of angiogenesis and mesenchymal stem cell expansion and differentiation. A recent study showed that CYR61 is expressed during fracture healing and suggested that CYR61 plays a significant role in cartilage and bone formation. The hypothesis of the present study was that decreased fixation stability, which leads to a delay in healing, would lead to reduced CYR61 protein expression in fracture callus. The aim of the study was to quantitatively analyze CYR61 protein expression, vascularization, and tissue differentiation in the osteotomy gap and relate to the mechanical fixation stability during the course of healing. A mid-shaft osteotomy of the tibia was performed in two groups of sheep and stabilized with either a rigid or semirigid external fixator, each allowing different amounts of interfragmentary movement. The sheep were sacrificed at 2, 3, 6, and 9 weeks postoperatively. The tibiae were tested biomechanically and histological sections from the callus were analyzed immunohistochemically with regard to CYR61 protein expression and vascularization. Expression of CYR61 protein was upregulated at the early phase of fracture healing (2 weeks), decreasing over the healing time. Decreased fixation stability was associated with a reduced upregulation of the CYR61 protein expression and a reduced vascularization at 2 weeks leading to a slower healing. The maximum cartilage callus fraction in both groups was reached at 3 weeks. However, the semirigid fixator group showed a significantly lower CYR61 immunoreactivity in cartilage than the rigid fixator group at this time point. The fraction of cartilage in the semirigid fixator group was not replaced by bone as quickly as in the rigid fixator group leading to an inferior histological and mechanical callus quality at 6 weeks and therefore to a slower healing. The results supply further evidence that CYR61 may serve as an important regulator of bone healing.

Although the sheep is a standard model for the analysis of biological healing processes after surgical intervention at the knee and shank, the in vivo tibio-femoral joint contact forces and the resulting musculoskeletal loading conditions have yet to be studied in detail.The three-dimensional kinematics of three Merino-mix sheep right hind limbs were recorded using reflective markers that were attached to Schanz’ screws, firmly anchored in the bone. This motion data was used together with the simultaneously measured external loading (ground reaction forces) to calculate the muscle and joint contact forces, as well as the internal loads within the bones. Whilst the motion mainly occurred in the sagittal plane, significant out of plane motion was observed, especially at the hip and knee joint (ab/adduction hip: 13°, knee: 10°; internal/external rotation hip: 12°, knee: 14°). The axial component of the tibio-femoral contact force was 2.1 times body weight (BW). The medio-lateral and anterior-posterior shear forces amounted to only 0.7 times BW. The loading in the diaphysis of the tibia under physiological musculoskeletal conditions was mainly axial compression (0.89 BW) together with only small shear forces (0.15 BW). The results of the musculoskeletal analyses presented here add to the understanding of the mechanical loading conditions in sheep. This expanded knowledge aids in the interpretation

Fracture healing requires a certain degree of mechanical stability and an adequate blood supply. The hypothesis of the present study was that increased interfragmentary shear leads to a reduced initial vascularization and prolonged healing. The aim of the study was to quantitatively analyze the histological appearance of vascularization and tissue differentiation with regard to fracture stability during the course of healing. A mid-shaft osteotomy of the tibia was performed in two groups of sheep and stabilized with either a rigid or semirigid external fixator, differing in bending stiffness. Interfragmentary movements and ground reaction forces were evaluated in vivo during a 9-week period. The sheep were sacrificed at 2, 3, 6, and 9 weeks postoperatively. The tibiae were tested biomechanically and histological sections from the callus were prepared for analysis of tissue differentiation and vascularization. Larger interfragmentary shear movements in the semirigid fixator group were associated with a reduced initial blood supply. At 6 weeks the semirigid fixator group showed a significantly lower percentage of mineralized bone and a higher amount of fibrous tissue leading to a significantly lower stiffness of the callus than the rigid fixator group. This initial delay in healing was compensated for in the later stages with the production of greater volumes of callus tissue so that both groups showed the same callus stiffness at 9 weeks. However, the rigid fixator group showed signs of the beginning of callus remodeling at the latest time points suggesting a faster bone healing. The results indicate the important role of the initial mechanical stability specifically in the vascularization of an osteosynthesis. Further studies should illustrate the precise role of mechanical conditions on the regulation of angiogenesis during early bone healing.

Pin loosening is a major complication in external fixation. Biological and mechanical conditions play an important role in the maintenance and enhancement of the implant-bone interface in fracture fixation. It is thought that biodegradable coatings may be capable of preventing pin track infection and pin loosening. The goal of this study was therefore to analyze the influence of a biodegradeable coating on the osseous integration of Schanz' screws during fracture treatment. Standardized osteotomies (3-mm fracture gap) of the right tibiae were performed on 16 sheep and stabilized by an AO mono-lateral external fixator. Additional, mechanically less loaded Schanz' screws were also mounted. All screws were randomly coated with biodegradable poly(D,L-lactide). The sheep were sacrificed after 9 weeks. All screws were removed and rolled on blood agar plates for microbiological analysis. Histological sections of the pin tracks were histochemically and morphometrically analyzed. Clinically, no signs of severe infection were visible. Microbiological analysis revealed 14.8% colonization by Staphylococcus aureus in the coated and 29% in the uncoated screws. Histomorphometry of the bone surrounding the Schanz' screws revealed that significantly more osseous integration had occurred on poly(D,L-lactide)-coated screws in the absence of bacterial colonization. Significantly more bone remodeling and a higher osteoclastic activity was seen near the screw-bone interface in the uncoated screw group. Up to a threefold increase in new bone formation and more severe remodeling was observed around the screw entry compared to the pin exit in all groups. Loaded screws showed significantly more callus formation around the exit sites than their less loaded counterparts. In the present study, poly(D,L-lactide) coating of Schanz' screws was found to enhance osseous integration in the absence of bacterial colonization in sheep by causing less cortical remodeling and less osteoclastic activity in the cortices compared to uncoated screws. Additionally, the coating appeared to reduce the instances of pin track infections. Mechanical loading showed an adverse effect on bone formation and remodeling. It has been shown that both biological and mechanical factors play an important role in the maintenance of osseous integrity of the pin-bone interface. Poly(D,L-lactide) coating of Schanz' screws does not prevent osseous destruction and severe bacterial colonization along the pin tracts, but can improve osseous integration of Schanz' screws in the absence of infection.

Locked intramedullary nailing and external fixation are alternatives for the stabilization of tibial shaft fractures. The goal of this study was to determine to what extent the mechanical conditions at the fracture site influence the healing process after unreamed tibial nailing compared to external fixation. A standardized tibial diastasis was stabilized with either a locked unreamed tibial nail or a monolateral fixator in a sheep model. Interfragmentary movements and ground reaction parameters were monitored in vivo throughout the healing period. After sacrifice, the tibiae were examined mechanically and histologically. Bending angles and axial torsion at the fracture site were larger in the nail group within the first five weeks post-operatively. Unlike the fixator group, the operated limb in the nail group did not return to full weight bearing during the treatment period. Mechanical and histomorphometrical observations showed significantly inferior bone healing in the nail group compared to the fixator group. In this study, unreamed nailing of a tibial diastasis did not provide rotational stability of the osteosynthesis and resulted in a significant delay in bone healing.

The aim of this study was to determine the ability of the following bone turnover markers to monitor the course of callus consolidation during bone healing: Carboxy-terminal propeptide of procollagen type I (PICP), skeletal alkaline phosphatase (sALP), and amino-terminal propeptide of type III procollagen (PIlINP). Since interfragmentary movements have been proven to possess the ability to document the progression of bone healing in experimental studies, correlations between bone turnover markers and interfragmentary movements in vivo were investigated. Therefore, two different types of osteosyntheses representing different mechanical situations at the fracture site were compared in an ovine osteotomy model. Blood samples were taken preoperatively and postoperatively in weekly intervals over a nine-week healing period. At the same intervals, interfragmentary movements were measured in all sheep. After nine weeks, animals were sacrificed and the tibiae were evaluated both mechanically and histologically. Wide interindividual ranges were observed for all bone turnover markers. The systemic PICP level did not increase with callus consolidation. The bone-healing model seemed to influence the systemic level of PIIINP and sALP but no general correlation between bone turnover markers and interfragmentary movements could be detected. No differences between the different types of osteosyntheses and thus the different mechanical situations were observed. All analyzed markers failed as general predictors for the course of callus consolidation during bone healing.

Schraubenkanal-Infektionen und Schraubenlockerungen sind wesentliche Komplikationen bei der Fixateur externe-Osteosynthese. In dieser Studie wurde der Einfluss einer Poly(D,L-laktid)-Beschichtung auf die ossare Integration der Schrauben wahrend der Frakturversorgung analysiert. An 16 Schafen wurden Osteotomien der rechten Tibia mit einem monolateralen Fixateur externe stabilisiert. Zusatzlich wurden mechanisch unbelastete Schanz' Schrauben eingebracht. Die Halfte der implantierten Schrauben wurde mit Poly(D,L-laktid) beschichtet. Nach neun Wochen wurden die Schrauben entnommen, auf Blutagar-Platten ausgerollt und mikrobiologisch untersucht. Es erfolgte die histologische Aufarbeitung der Pintrakts und eine anschliesende histochemische und histomorphometrische Analyse. Obwohl klinisch keine Anzeichen fur Infektionen beobachtet wurden, zeigte die mikrobiologische Bewertung bei 15 % der beschichteten Schrauben versus 29 % der unbeschichteten Schrauben eine Kontamination durch Staphylococcus aureus. Die histologische Auswertung zeigte, dass beschichtete Schrauben eine bessere Osteointegritat (p=0,0006) als unbeschichtete Implantate aufwiesen. In der Histomorphometrie wurde in der Gruppe der unbeschichteten Schrauben ein hoheres knochernes Remodeling (p = 0,006) und eine hohere Osteoklastenaktivitat (p = 0,019) festgestellt. Belastete Schrauben zeigten mehr Kallus am Schraubenaustritt (p = 0,048). Eine Poly(D,L-laktid)-Beschichtung bei Schanz' Schrauben scheint die ossare Integration durch eine Verminderung des kortikalen Remodelings und der Osteoklastenaktivitat zu verbessern. Improvement of bony integration of Schanz’ screws by means of a Poly(D,L-laktid) coating Pin track infections and pin loosening are major complications in fracture treatment by means of external fixation. In this study, the influence of Poly(D,L-lactid)-coating on osseous integration of Schanz screws was analysed during bone healing. In sixteen sheep, osteotomies of the right tibia were stabilized by means of monolateral external fixators. In addition, mechanically unloaded Schanz' screws were inserted. One half of the implanted screws was coated with Poly(D,L-lactid). After nine weeks in vivo, the screws were extracted, rolled on blood agar plates and microbiologically analysed, followed by histological, immunhistochemical and histomorphometrical analyses of the pin tracks. Even though no clinical signs of infection were observed, micro biological evaluation indicated a contamination by Staphylococcus aureus of 15 % in the coated screws and of 29 % in the uncoated screws. The histological analyses showed an improved osseous interagtion in the coated screws (p=0,0006) compared to the uncoated implants. In histomorphometry, the group of the uncoated screws showed an increased bone remodelling (p = 0,006) and an increased osteoclast activity (p = 0,019). Mechanically loaded screws had increased callus formation at the pin exit site (p = 0,048). The poly(D,L-lactid) coating of Schanz' screws seems to enhance the osseous integration by reducing cortical remodelling and osteoclast activity.

Mechanical properties and good biocompatibility of zirconium and some of its alloys focus these materials as good candidates for biomedical applications. The attractive in vivo performance of zirconium is mainly due to the presence of a protective oxide layer. In this preliminary study, surface modification of pure zirconium was made by anodisation in acidic media at low potentials, enhancing the barrier protection given by the oxides and the osseointegration. Electrochemical and SEM (scanning electron microscopy) assays were done in commercially pure zirconium cylinders and two surface conditions were compared: pure and zirconium anodised at 30V. The in vivo assays were held in a tibia rat model. The histological features and fluorochrome labelling changes of newly bone formed around implants were evaluated on the non-decalcified sections 63 days after surgery. Electrochemical and SEM assays showed that anodisation treatment would increased the barrier effect over the material and the in vivo assays showed a continuous newly bone formation around the implant with a different amount of osteocytes in their lacunaes depending the region. There was no significant change in bone thickness around both kinds of implants but conversely a significant increase in the mineralization apposition rate was determined for the anodised condition suggesting that anodisation treatment would stimulate and assist to the osseointegration process. We conclude that anodisation treatment at 30 V can stimulate the implant fixation in a rat model and this study would propose the study of zirconium as candidate material for permanent implant. Fil: Katunar, Maria Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigación En Ciencia y Tecnología de Materiales (i); Argentina. Universidad Nacional de Mar del Plata. Facultad de Ingeniería; Argentina Fil: Gomez Sanchez, Andrea Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigación En Ciencia y Tecnología de Materiales (i); Argentina. Universidad Nacional de Mar del Plata. Facultad de Ingeniería; Argentina Fil: Ballarre, Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigación En Ciencia y Tecnología de Materiales (i); Argentina. Universidad Nacional de Mar del Plata. Facultad de Ingeniería; Argentina Fil: Baca, Matías. Hospital Interzonal General de Agudos ‘‘Oscar Alende’’. Mar del Plata; Argentina Fil: Vottola, Carlos. Hospital Interzonal General de Agudos ‘‘Oscar Alende’’; Argentina Fil: Orellano, Juan C.. Hospital Interzonal General de Agudos ‘‘Oscar Alende’’. Mar del Plata; Argentina Fil: Schell, Hanna. Universitatsmedizin Berlin; Alemania Fil: Duffo, Gustavo Sergio. Comision Nacional de Energia Atomica. Gerencia D/area de Energia Nuclear. Unidad de Actividad de Materiales (cac); Argentina. Universidad Nacional de San Martín; Argentina Fil: Cere, Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigación En Ciencia y Tecnología de Materiales (i); Argentina. Universidad Nacional de Mar del Plata. Facultad de Ingeniería; Argentina

It is widely accepted, that mechanical stability of monolateral external fixators has an effect on the healing progress and clinical outcome. Recent analytical analyses suggest an influence of the mounting plane on fracture gap movement in sheep. Goal was to determine interfragmentary movement for different mounting planes of external fixators and correlate these to analytical predictions of gap movements from a computer model.

Fracture healing is a regenerative process in which bone is restored without scar tissue formation. The healing cascade initiates with a cycle of inflammation, cell migration, proliferation and differentiation. Immune cells invade the fracture site immediately upon bone damage and contribute to the initial phase of the healing process by recruiting accessory cells to the injury site. However, little is known about the role of the immune system in the later stages of fracture repair, in particular, whether lymphocytes participate in soft and hard callus formation. In order to answer this question, we analyzed femoral fracture healing in mice by confocal microscopy. Surprisingly, after the initial inflammatory phase, when soft callus developed, T and B cells withdrew from the fracture site and were detectable predominantly at the femoral neck and knee. Thereafter lymphocytes massively infiltrated the callus region (around day 14 after injury), during callus mineralization. Interestingly, lymphocytes were not found within cartilaginous areas of the callus but only nearby the newly forming bone. During healing B cell numbers seemed to exceed those of T cells and B cells progressively underwent effector maturation. Both, osteoblasts and osteoclasts were found to have direct cell-cell contact with lymphocytes, strongly suggesting a regulatory role of the immune cells specifically in the later stages of fracture healing.

Experimental data indicate that hypercapnic adidosis has anti-inflammatory effects. These anti-inflammatory effects may even be a beneficial property in case of low tidal volume ventilation with consecutive hypercapnic acidosis. It is unclear whether these anti-inflammatory effects predominate in critically ill patients who suffer from multiple pro- and anti-inflammatory insults like extracorporeal organ support (pro-inflammatory), metabolic acidosis (pro- and anti-inflammatory), as well as hypoxia (pro-inflammatory). Eighteen pigs were randomized into three groups, mechanically ventilated and connected to a continuous veno-venous hemofiltration (CVVH) as pro-inflammatory insult. A reference group with normal acid-base state obtained normoventilation; a normoxemic acidemia group obtained normoxemic, mixed acidemia due to infusion of lactic and hyperchloremic acid and low tidal volume ventilation, and in a hypoxemic acidemia group the mixed acidemia was paralleled by hypoxemia. Lung histology including pulmonary leukocyte invasion, blood gases, blood cell counts, and hemodynamics were examined. The histological examination of the lungs of acidemic pigs showed a suppressed invasion of leukocytes and thinner alveolar walls compared with normoventilated and with hypoxemic pigs. Enhanced congestion and alveolar red blood cells (RBCs) combined with an increase of the pulmonary artery pressure were observed in acidemic pigs in comparison with the reference group. Normoxemic acidemia reduced the pro-inflammatory reaction to the CVVH and mechanical ventilation in the ventilated lung areas in the form of pulmonary leukocyte invasion. However, this did not result in reduced scores for lung injury. Instead, an increased score for criteria which represent lung injury (congestion and alveolar RBCs) was observed in acidemic pigs.

The reconstruction of large defects (>10 mm) in humans usually relies on bone graft transplantation. Limiting factors include availability of graft material, comorbidity, and insufficient integration into the damaged bone. We compare the gold standard autograft with biodegradable composite scaffolds consisting of medical-grade polycaprolactone and tricalcium phosphate combined with autologous bone marrow-derived mesenchymal stem cells (MSCs) or recombinant human bone morphogenetic protein 7 (rhBMP-7). Critical-sized defects in sheep - a model closely resembling human bone formation and structure - were treated with autograft, rhBMP-7, or MSCs. Bridging was observed within 3 months for both the autograft and the rhBMP-7 treatment. After 12 months, biomechanical analysis and microcomputed tomography imaging showed significantly greater bone formation and superior strength for the biomaterial scaffolds loaded with rhBMP-7 compared to the autograft. Axial bone distribution was greater at the interfaces. With rhBMP-7, at 3 months, the radial bone distribution within the scaffolds was homogeneous. At 12 months, however, significantly more bone was found in the scaffold architecture, indicating bone remodeling. Scaffolds alone or with MSC inclusion did not induce levels of bone formation comparable to those of the autograft and rhBMP-7 groups. Applied clinically, this approach using rhBMP-7 could overcome autograft-associated limitations.

Critical or delayed bone healing in rat osteotomy (OT) models is mostly achieved through large defects or instability. We aimed to design a rat OT model for impaired bone healing based on age, gender and parity. The outcome should be controllable through variations of the haematoma in the OT including a bone morphogenetic protein (BMP) 2 guided positive control. Using external fi xation to stabilise femoral a 2 mm double OT in 12 month old, female Sprague Dawley rats after a minimum of 3 litters healing was characterised following in situ haematoma formation (ISH-group)), transplantation of a BMP charged autologous blood clot (BMP-group) and the artifi cial blood clot only (ABC-group) into the OT-gap. In vivo micro-computer tomography (μCT) scans were performed after 2, 4 and 6 weeks. After 6 weeks specimens underwent histological analyses. In μCT examinations and histological analyses no bony bridging was observed in all but one animal in the ISHgroup. In the BMP group complete bridging was achieved in all animals. The ABC-group showed less mineralised tissue formation and smaller bridging scores during the course of healing than the ISH-group. In this pilot study we introduce a model for impaired bone healing taking the major biological risk factors into account. We could show that the in situ fracture haematoma is essential for bone regeneration. Using BMP as a positive control the presented experimental setup can serve to evaluate innovative therapeutical concepts in long bone application.

Critical-sized bone defect regeneration is a remaining clinical concern. Numerous scaffold-based strategies are currently being investigated to enable in vivo bone defect healing. However, a deeper understanding of how a scaffold influences the tissue formation process and how this compares to endogenous bone formation or to regular fracture healing is missing. It is hypothesized that the porous scaffold architecture can serve as a guiding substrate to enable the formation of a structured fibrous network as a prerequirement for later bone formation. An ovine, tibial, 30-mm critical-sized defect is used as a model system to better understand the effect of the scaffold architecture on cell organization, fibrous tissue, and mineralized tissue formation mechanisms in vivo. Tissue regeneration patterns within two geometrically distinct macroscopic regions of a specific scaffold design, the scaffold wall and the endosteal cavity, are compared with tissue formation in an empty defect (negative control) and with cortical bone (positive control). Histology, backscattered electron imaging, scanning small-angle X-ray scattering, and nanoindentation are used to assess the morphology of fibrous and mineralized tissue, to measure the average mineral particle thickness and the degree of alignment, and to map the local elastic indentation modulus. The scaffold proves to function as a guiding substrate to the tissue formation process. It enables the arrangement of a structured fibrous tissue across the entire defect, which acts as a secondary supporting network for cells. Mineralization can then initiate along the fibrous network, resulting in bone ingrowth into a critical-sized defect, although not in complete bridging of the defect. The fibrous network morphology, which in turn is guided by the scaffold architecture, influences the microstructure of the newly formed bone. These results allow a deeper understanding of the mode of mineral tissue formation and the way this is influenced by the scaffold architecture. © 2012 American Society for Bone and Mineral Research.

Purpose It is unknown what causes donor site morbidity following the osteochondral autograft transfer procedure or how donor sites heal. Contact pressure and edge loading at donor sites may play a role in the healing process. It was hypothesized that an artificially created osteochondral defect in a weightbearing area of an ovine femoral condyle will cause osseous bridging of the defect from the upper edges, resulting in incomplete and irregular repair of the subchondral bone plate. Methods To simulate edge loading, large osteochondral defects were created in the most unfavourable weightbearing area of 24 ovine femoral condyles. After killing at 3 and 6 months, osteochondral defects were histologically and histomorphometrically evaluated with specific attention to subchondral bone healing and subchondral bone plate restoration. Results Osteochondral defect healing showed progressive osseous defect bridging by sclerotic circumferential bone apposition. Unfilled area decreased significantly from 3 to 6 months (P = 0.004), whereas bone content increased (n.s.). Complete but irregular subchondral bone plate restoration occurred in ten animals. In fourteen animals, an incomplete subchondral bone plate was found. Further common findings included cavitary lesion formation, degenerative cartilage changes and cartilage and subchondral bone collapse. Conclusions Osteochondral defect healing starts with subchondral bone plate restoration. However, after 6 months, incomplete or irregular subchondral bone plate restoration and subsequent failure of osteochondral defect closure is common. Graft harvesting in the osteochondral autograft transfer procedure must be viewed critically, as similar changes are also present in humans. Level of evidence Prognostic study, Level III.

During hematoma formation following injury, an inflammatory reaction ensues as an initial step in the healing process. As granulation tissue matures, revascularization is a prerequisite for successful healing. The hypothesis of this study was that scarless tissue reconstitution in the regenerative bone healing process is dependent on a balanced immune reaction that initiates revasculatory steps. To test this hypothesis, cellular composition and expression profiles of a bone hematoma (regenerative, scarless) was compared with a muscle soft tissue hematoma (healing with a scar) in a sheep model. Upregulation of regulatory T helper cells and anti-inflammatory cytokine expression (IL-10) coincided with an upregulation of angiogenic factors (HIF1α and HIF1α regulated genes) in the regenerative bone hematoma but not in the soft tissue hematoma. These results indicate that the timely termination of inflammation and early onset of revascularization are interdependent and essential for a regenerative healing process. Prolonged pro-inflammatory signaling occurring in a delayed bone-healing model supports the finding that timely termination of inflammation furthers the regenerative process. Differing cellular compositions are due to different cell sources invading the hematoma, determining the ensuing cytokine expression profile and thus paving the path for regenerative healing in bone or the formation of scar tissue in muscle injury.