Elmar Willbold

Magnesium hydroxide temporarily enhancing osteoblast activity and decreasing the osteoclast number in peri-implant bone remodelling.

Repeated observations of enhanced bone growth around various degradable magnesium alloys in vivo raise the question: what is the major mutual origin of this biological stimulus? Several possible origins, e.g. the metal surface properties, electrochemical interactions and biological effects of alloying elements, can be excluded by investigating the sole bone response to the purified major corrosion product of all magnesium alloys, magnesium hydroxide (Mg(OH)(2)). Isostatically compressed cylinders of pure Mg(OH)(2) were implanted into rabbit femur condyles for 2-6 weeks. We observed a temporarily increased bone volume (BV/TV) in the vicinity of Mg(OH)(2) at 4 weeks that returned to a level that was equal to the control at 6 weeks. The osteoclast surface (OcS/BS) was significantly reduced during the first four weeks around the Mg(OH)(2) cylinder, while an increase in osteoid surface (OS/BS) was observed at the same time. At 6 weeks, the OcS/BS adjacent to the Mg(OH)(2) cylinder was back within the same range of the control. The mineral apposition rate (MAR) was extensively enhanced until 4 weeks in the Mg(OH)(2) group before matching the control. Thus, the enhanced bone formation and temporarily decreased bone resorption resulted in a higher bone mass around the slowly dissolving Mg(OH)(2) cylinder. These data support the hypothesis that the major corrosion product Mg(OH)(2) from any magnesium alloy is the major origin of the observed enhanced bone growth in vivo. Further studies have to evaluate if the enhanced bone growth is mainly due to the local magnesium ion concentration or the local alkalosis accompanying the Mg(OH)(2) dissolution.

Fast escape of hydrogen from gas cavities around corroding magnesium implants.

Magnesium materials are of increasing interest in the development of biodegradable implants as they exhibit properties that make them promising candidates. However, the formation of gas cavities after implantation of magnesium alloys has been widely reported in the literature. The composition of the gas and the concentration of its components in these cavities are not known as only a few studies using non-specific techniques were done about 60 years ago. Currently many researchers assume that these cavities contain primarily hydrogen because it is a product of magnesium corrosion in aqueous media. In order to clearly answer this question we implanted rare earth-containing magnesium alloy disks in mice and determined the concentration of hydrogen gas for up to 10 days using an amperometric hydrogen sensor and mass spectrometric measurements. We were able to directly monitor the hydrogen concentration over a period of 10 days and show that the gas cavities contained only a low concentration of hydrogen gas, even shortly after formation of the cavities. This means that hydrogen must be exchanged very quickly after implantation. To confirm these results hydrogen gas was directly injected subcutaneously. Most of the hydrogen gas was found to exchange within 1h after injection. Overall, our results disprove the common misbelief that these cavities mainly contain hydrogen and show how quickly this gas is exchanged with the surrounding tissue.

In vitro and in vivo evaluation of biodegradable, open-porous scaffolds made of sintered magnesium W4 short fibres

A cytocompatible and biocompatible, degradable, open-porous, mechanically adaptable metal scaffold made of magnesium alloy W4 melt-extracted short fibres was fabricated by liquid phase sintering. Cylindrical samples (3×5 mm) of sintered W4 short fibres were evaluated under in vitro (L929, HOB, eudiometer, weight loss) and in vivo conditions (rabbits: 6 and 12 weeks). The in vitro corrosion environment (e.g., temperature, flow, composition of corrosion solution, exposure time) significantly influenced the corrosion rates of W4 scaffolds compared with corrosion in vivo. Corrosion rates under cell culture conditions for 72 h varied from 1.05 to 3.43 mm y(-1) depending on the media composition. Corrosion rates measured in eudiometric systems for 24 h were ~24-27 times higher (3.88-4.43 mm y(-1)) than corrosion in vivo after 6 weeks (0.16 mm y(-1)). Moreover, it was found that the cell culture media composition significantly influences the ionic composition of the extract by selectively dissolving ions from W4 samples or their corrosion products. A pilot in vivo study for 6 and 12 weeks demonstrated active bone remodelling, no foreign body reaction and no clinical observation of gas formation during W4 scaffold implantation. Long-term in vivo studies need to be conducted to prove complete degradation of the W4 scaffold and total replacement by the host tissue.

Simultaneous regeneration of articular cartilage and subchondral bone induced by spatially presented TGF-beta and BMP-4 in a bilayer affinity binding system

Subchondral defect repair is a multitask challenge requiring the simultaneous regeneration of cartilage and bone. Herein, we describe the features of a hydrogel system designed to simultaneously induce the endogenous regeneration of hyaline cartilage and subchondral bone. The system was constructed as two layers, spatially presenting the chondroinductive transforming growth factor-β1 (TGF-β1) in one layer and the osteoinductive bone morphogenetic protein-4 (BMP-4) in a second layer, via affinity binding to the matrix. Human mesenchymal stem cells seeded in the bilayer system differentiated into chondrocytes and osteoblasts in the respective layers, confirming the spatial presentation and prolonged activity of TGF-β1 and BMP-4. Administration of the bilayer system with affinity-bound TGF-β1 and BMP-4 (with no cells) into a subchondral defect in rabbits induced endogenous regeneration of articular cartilage and the subchondral bone underneath within 4weeks. Cartilage extracellular matrix proteoglycans were found in the top layer, with no mineralization, whereas the layer underneath consisted of newly formed woven bone. The results indicate that stem cells migrating into the defect are able to sense the biological cues spatially presented in the hydrogel and respond by differentiation into the appropriate cell lineage. The strategy has a real translational potential for repairing osteochondral defects in humans as it is acellular and can be implanted via a minimally invasive method.

Effect of the addition of low rare earth elements (lanthanum, neodymium, cerium) on the biodegradation and biocompatibility of magnesium

Rare earth elements are promising alloying element candidates for magnesium alloys used as biodegradable devices in biomedical applications. Rare earth elements have significant effects on the high temperature strength as well as the creep resistance of alloys and they improve magnesium corrosion resistance. We focused on lanthanum, neodymium and cerium to produce magnesium alloys with commonly used rare earth element concentrations. We showed that low concentrations of rare earth elements do not promote bone growth inside a 750 μm broad area around the implant. However, increased bone growth was observed at a greater distance from the degrading alloys. Clinically and histologically, the alloys and their corrosion products caused no systematic or local cytotoxicological effects. Using microtomography and in vitro experiments, we could show that the magnesium-rare earth element alloys showed low corrosion rates, both in in vitro and in vivo. The lanthanum- and cerium-containing alloys degraded at comparable rates, whereas the neodymium-containing alloy showed the lowest corrosion rates.

Biocompatibility of rapidly solidified magnesium alloy RS66 as a temporary biodegradable metal

Biodegradable magnesium-based alloys are very promising materials for temporary implants. However, the clinical use of magnesium-based alloys is often limited by rapid corrosion and by insufficient mechanical stability. Here we investigated RS66, a magnesium-based alloy with extraordinary physicochemical properties of high tensile strength combined with a high ductility and a homogeneous grain size of ~1 μm which was obtained by rapid solidification processing and reciprocal extrusion. Using a series of in vitro and in vivo experiments, we analyzed the biodegradation behavior and the biocompatibility of this alloy. In vitro, RS66 had no cytotoxic effects in physiological concentrations on the viability and the proliferation of primary human osteoblasts. In vivo, RS66 cylinders were implanted into femur condyles, under the skin and in the muscle of adult rabbits and were monitored for 1, 2, 3, 4 and 8 weeks. After explantation, the RS66 cylinders were first analyzed by microtomography to determine the remaining RS66 alloy and calculate the corrosion rates. Then, the implantation sites were examined histologically for healing processes and foreign body reactions. We found that RS66 was corroded fastest subcutaneously followed by intramuscular and bony implantation of the samples. No clinical harm with transient gas cavities during the first 6 weeks in subcutaneous and intramuscular implantation sites was observed. No gas cavities were formed around the implantation site in bone. The corrosion rates in the different anatomical locations correlated well with the local blood flow prior to implantation. A normal foreign body reaction occurred in all tissues. Interestingly, no enhanced bone formation could be observed around the corroding samples in the condyles. These data show that RS66 is biocompatible, and due to its interesting physicochemical properties, this magnesium alloy is a promising material for biodegradable implants.

Histology and research at the hard tissue–implant interface using Technovit 9100 New embedding technique

Calcified tissues, like bones and teeth, are among the most challenging tissues for histological research. However, especially with respect to dental or orthopaedic research, powerful histological techniques are necessary to study pathological conditions or traumatic injuries, and to investigate the molecular and cellular mechanisms of regeneration processes and functional recovery. The situation is even more complicated in orthopaedic research because here metallic implants or other devices made of various materials are often present, and the hard tissue-implant interface is of crucial interest in both biocompatibility and functional recovery research. After the cutting-grinding technique, embedding in technical resins is the most promising approach. Here we describe an optimized and standardized embedding and cutting technique using Technovit 9100 New. Using this technique, we are able to perform enzyme histochemistry, immunohistochemistry, a great variety of classical histological stains and even in situ hybridization.

Postnatal maturation of tendon, cruciate ligament, meniscus and articular cartilage: a histological study in sheep.

Orthopaedic basic science data on immature skeletons are rare in the literature. Since the number of knee injuries in young humans is steadily increasing, studies on immature animals such as sheep, which can be used as model systems are becoming more and more important. However, no baseline data are available on physiologic and morphologic changes during growth in the relevant tissues. In the present study, histomorphometric changes in the tendon of the musculus flexor digitalis superficialis, the cranial cruciate ligament, the medial meniscus and the articular cartilage of the medial femoral condyle were identified in sheep between the ages of 1 and 40 weeks postnatally. Profound changes in tissue composition during growth could be observed. A high cellularity in the early postnatal period decreases to a constant lower level after 18 weeks. Similar changes during postnatal growth could be observed for blood vessel density. Also, staining of alpha-smooth muscle actin (SMA) and vascular endothelial growth factor (VEGF) steadily decreased. In contrast, the number of components of extracellular matrix steadily increased in all tissues. The age of 18 weeks seems to be a threshold after which the tissue composition of the observed structures remains constant in this species.

Mesenchymal Stem Cell‐Dependent Formation of Heterotopic Tendon‐Bone Insertions (Osteotendinous Junctions)

Ligament‐to‐bone and tendon‐to‐bone interfaces (entheses, osteotendinous junctions [OTJs]) serve to dissipate stress between soft tissue and bone. Surgical reconstruction of these interfaces is an issue of considerable importance as they are prone to injury and the integration of bone and tendon/ligament is in general not satisfactory. We report here the stem cell‐dependent spontaneous formation of fibrocartilaginous and fibrous entheses in heterotopic locations of the mouse if progenitors possess a tenogenic and osteo‐/chondrogenic capacity. This study followed the hypothesis that enhanced Bone Morphogenetic Protein (BMP)‐signaling in adult mesenchymal stem cells that are induced for tendon formation may overcome the tendon‐inherent interference with bone formation and may thus allow the stem cell‐dependent formation of tendon‐bone interfaces. The tenogenic and osteo‐/chondrogenic competence was mediated by the adeno‐ and/or lentiviral expression of the biologically active Smad8 signaling mediator (Smad8ca) and of Bone Morphogenetic Protein 2 (BMP2). Modified mesenchymal progenitors were implanted in subcutaneous or intramuscular sites of the mouse. The stem cell‐dependent enthesis formation was characterized histologically by immunohistological approaches and by in situ hybridization. Transplantation of modified murine stem cells resulted in the formation of tendinous and osseous structures exhibiting fibrocartilage‐type OTJs, while, in contrast, the viral modification of primary human bone marrow‐derived mesenchymal stromal/stem cells showed evidence of fibrous tendon‐bone interface formation. Moreover, it could be demonstrated that Smad8ca expression alone was sufficient for the formation of tendon/ligament‐like structures. These findings may contribute to the establishment of stem cell‐dependent regenerative therapies involving tendon/ligaments and to the improvement of the insertion of tendon grafts at bony attachment sites, eventually. STEM CELLS 2010; 28:1590–1601.

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

PURPOSE It was our aim to establish an animal model and to investigate the tendon graft-to-bone and physis healing process in skeletally immature sheep after reconstruction of the anterior cruciate ligament (ACL). METHODS Thirty-two immature sheep aged 4 months underwent a fully transphyseal ACL reconstruction by use of a soft-tissue graft. The animals were subsequently killed after 3, 6, 12, and 24 weeks and analyzed histologically and biomechanically. RESULTS There was a transient hypertrophy of the physis tissue at the passing site of the graft. Anchoring Sharpey-like fibers evolved as early as 3 weeks after surgery. A strong expression of collagen III messenger ribonucleic acid within the first 6 weeks preceded this anchoring process. The maximum load to failure of the tendon graft in the reconstructed knees initially decreased to 37.8 +/- 17.8 N after 3 weeks and was restored to 522.9 +/- 113 N after 24 weeks. Tendon graft stiffness was restored to 86% when compared with the control knees. CONCLUSIONS The early anchoring by Sharpey fibers was found at 3 weeks with continued maturation to 24 weeks. This development of anchoring fibers corresponded to that of biomechanical strength, starting with 5% of the normal knee at 3 weeks and then 15.2% at 6 weeks, 41.2% at 12 weeks, and 69% at 24 weeks. Tendon graft-to-bone and physis healing in skeletally immature sheep is further characterized by a transient hypertrophy of the physis cartilage. The physis recovers well from the trauma of drilling and placement of a soft-tissue graft. The early development of Sharpey-like fibers results in a solid integration of the graft into bone in a timely manner. CLINICAL RELEVANCE ACL reconstruction in skeletally immature individuals is still controversial. This study describes in detail the histologic and biomechanical stages of tendon graft healing to the bone and physis. These data enrich the existing knowledge of previous studies in adult sheep and may provide a basis for further research in the controversial field of ACL reconstruction during growth.