Showing papers by "Georg N. Duda published in 2011"

Fracture healing is accelerated in the absence of the adaptive immune system.

Abstract: Fracture healing is a unique biologic process starting with an initial inflammatory response. As in other regenerative processes, bone and the immune system interact closely during fracture healing. This project was aimed at further elucidating how the host immune system participates in fracture healing. A standard closed femoral fracture was created in wild-type (WT) and recombination activating gene 1 knockout (RAG1(-/-)) mice lacking the adaptive immune system. Healing was investigated using micro-computed tomography (µCT), biomechanical testing, and histologic and mRNA expression analyses. Biomechanical testing demonstrated a significantly higher torsional moment on days 14 and 21 in the RAG1(-/-) mice compared to the WT group. µCT evaluation of RAG1(-/-) specimens showed earlier mineralization and remodeling. Histologically, endochondral ossification and remodeling were accelerated in the RAG1(-/-) compared with the WT mice. Histomorphometric analysis on day 7 showed a significantly higher fraction of bone and a significantly lower fraction of cartilage in the callus of the RAG1(-/-) mice than in the WT mice. Endochondral ossification was accelerated in the RAG1(-/-) mice. Lymphocytes were present during the physiologic repair process, with high numbers in the hematoma on day 3 and during formation of the hard callus on day 14 in the WT mice. Expression of inflammatory cytokines was reduced in the RAG1(-/-) mice. In contrast, expression of anti-inflammatory interleukin 10 (IL-10) was strongly upregulated in RAG1(-/-) mice, indicating protective effects. This study revealed an unexpected phenotype of enhanced fracture healing in RAG1(-/-) mice, suggesting detrimental functions of lymphocytes on fracture healing. The shift from proinflammatory to anti-inflammatory cytokines suggests that immunomodulatory intervention strategies that maximise the regenerative and minimize the destructive effects of inflammation may lead to enhanced fracture repair.

Human Early Fracture Hematoma Is Characterized by Inflammation and Hypoxia

Abstract: Background An effective immune system, especially during the inflammatory phase, putatively influences the quality and likelihood of bone healing. If and how this is reflected within the initial fracture hematoma is unclear.

Custom-made composite scaffolds for segmental defect repair in long bones

Abstract: Current approaches for segmental bone defect reconstruction are restricted to autografts and allografts which possess osteoconductive, osteoinductive and osteogenic properties, but face significant disadvantages. The objective of this study was to compare the regenerative potential of scaffolds with different material composition but similar mechanical properties to autologous bone graft from the iliac crest in an ovine segmental defect model. After 12 weeks, in vivo specimens were analysed by X-ray imaging, torsion testing, micro-computed tomography and histology to assess amount, strength and structure of the newly formed bone. The highest amounts of bone neoformation with highest torsional moment values were observed in the autograft group and the lowest in the medical grade polycaprolactone and tricalcium phosphate composite group. The study results suggest that scaffolds based on aliphatic polyesters and ceramics, which are considered biologically inactive materials, induce only limited new bone formation but could be an equivalent alternative to autologous bone when combined with a biologically active stimulus such as bone morphogenetic proteins.

CD73 and CD29 concurrently mediate the mechanically induced decrease of migratory capacity of mesenchymal stromal cells.

Abstract: The assumption that mesenchymal stromal cell (MSC)based-therapies are capable of augmenting physiological regeneration processes has fostered intensive basic and clinical research activities. However, to achieve sustained therapeutic success in vivo, not only the biological, but also the mechanical microenvironment of MSCs during these regeneration processes needs to be taken into account. This is especially important for e.g., bone fracture repair, since MSCs present at the fracture site undergo signifi cant biomechanical stimulation. This study has therefore investigated cellular characteristics and the functional behaviour of MSCs in response to mechanical loading. Our results demonstrated a reduced expression of MSC surface markers CD73 (ecto-5’-nucleotidase) and CD29 (integrin β1) after loading. On the functional level, loading led to a reduced migration of MSCs. Both effects persisted for a week after the removal of the loading stimulus. Specifi c inhibition of CD73/CD29 demonstrated their substrate dependent involvement in MSC migration after loading. These results were supported by scanning electron microscopy images and phalloidin staining of actin fi laments displaying less cell spreading, lamellipodia formation and actin accumulations. Moreover, focal adhesion kinase and Src-family kinases were identifi ed as candidate downstream targets of CD73/CD29 that might contribute to the mechanically induced decrease in MSC migration. These results suggest that MSC migration is controlled by CD73/CD29, which in turn are regulated by mechanical stimulation of cells. We therefore speculate that MSCs migrate into the fracture site, become mechanically entrapped, and thereby accumulate to fulfi l their regenerative functions.

The effect of design parameters of dynamic pedicle screw systems on kinematics and load bearing: an in vitro study.

Abstract: As an alternative treatment for chronic back pain due to disc degeneration motion preserving techniques such as posterior dynamic stabilization (PDS) has been clinically introduced, with the intention to alter the load transfer and the kinematics at the affected level to delay degeneration. However, up to the present, it remains unclear when a PDS is clinically indicated and how the ideal PDS mechanism should be designed to achieve this goal. Therefore, the objective of this study was to compare different PDS devices against rigid fixation to investigate the biomechanical impact of PDS design on stabilization and load transfer in the treated and adjacent cranial segment. Six human lumbar spine specimens (L3–L5) were tested in a spine loading apparatus. In vitro flexibility testing was performed by applying pure bending moments of 7.5 Nm without and with additional preload of 400 N in the three principal motion planes. Four PDS devices, “DYN” (Dynesys®, Zimmer GmbH, Switzerland), “DSS™” (Paradigm Spine, Wurmlingen, Germany), and two prototypes of dynamic rods, “LSC” with a leaf spring, and “STC” with a spring tube (Aesculap AG, Tuttlingen, Germany), were tested in comparison to a rigid fixation device S4 (Aesculap AG, Tuttlingen, Germany) “RIG”, to the native situation “NAT” and to a defect situation “DEF” of the specimens. The instrumented level was L4–L5. The tested PDS devices comprising a stiffness range for axial stiffness of 10 N/mm to 230 N/mm and for bending stiffness of 3 N/mm to 15 N/mm. Range of motion (ROM), neutral zone (NZ), and intradiscal pressure (IDP) were analyzed for all instrumentation steps and load cases of the instrumented and non-instrumented level. In flexion, extension, and lateral bending, all systems, except STC, showed a significant reduction of ROM and NZ compared to the native situation (p 0.1). In axial rotation, only DSS and STC reduced the ROM significantly (p 0.05). A correlation was found between axial stiffness and intersegmental stabilization in the sagittal and frontal plane, but not in the transversal plane where intersegmental stabilization is mainly governed by the systems’ ability to withstand shear loads. Furthermore, we observed the systems’ capacity to reduce IDP in the treated segment. The adjacent segment does not seem to be affected by the stiffness of the fixation device under the described loading conditions.

Composite transcriptome assembly of RNA-seq data in a sheep model for delayed bone healing

Abstract: Background The sheep is an important model organism for many types of medically relevant research, but molecular genetic experiments in the sheep have been limited by the lack of knowledge about ovine gene sequences.

A 5-mm femoral defect in female but not in male rats leads to a reproducible atrophic non-union.

Abstract: 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.

Time course of skeletal muscle regeneration after severe trauma.

Abstract: Background and purpose Animal models of skeletal muscle injury should be thoroughly described and should mimic the clinical situation. We established a model of a critical size crush injury of the ...

Spatial-Temporal Mapping of Bone Structural and Elastic Properties in a Sheep Model Following Osteotomy

Abstract: The course of bone healing in animal models is conventionally monitored by morphologic approaches, which do not allow the determination of the material properties of the tissues involved. Mechanical characterization techniques are either dedicated to the macroscopic evaluation of the entire organ or to the microscopic evaluation of the tissue matrix. The latter provides insight to regionally specific alterations at the tissue level in the course of healing. In this study, quantitative scanning acoustic microscopy was used at 50 MHz to investigate microstructural and elastic alterations of mineralized callus and cortical tissue after transverse osteotomy in sheep tibiae. Analyses were performed after 2, 3, 6 and 9 weeks of consolidation with stabilization by either a rigid or a semi-rigid external fixator. Increased stiffness and decreased porosity were observed in the callus tissue over the course of the healing process, which was dependent on the fixator type. In the adjacent cortical tissue, stiffness decreased during the first 3 weeks. Cortical porosity increased over time but the time-dependence was different between the two fixator types. The changes of stiffness of cortical and callus tissues were measured with respect to the distance to the periosteal cortex-callus boundary. Stiffness of cortex and callus tissue smoothly decreased as a function of the distance from the inner cortical region. The data obtained in this study can help to understand the processes involved in tissue maturation during endogenous bone healing. (E-mail: kay.raum@charite.de )

Time kinetics of bone defect healing in response to BMP-2 and GDF-5 characterised by in vivo biomechanics.

Abstract: This study reports that treatment of osseous defects with different growth factors initiates distinct rates of repair. We developed a new method for monitoring the progression of repair, based upon measuring the in vivo mechanical properties of healing bone. Two different members of the bone morphogenetic protein (BMP) family were chosen to initiate defect healing: BMP-2 to induce osteogenesis, and growth-and-differentiation factor (GDF)-5 to induce chondrogenesis. To evaluate bone healing, BMPs were implanted into stabilised 5 mm bone defects in rat femurs and compared to controls. During the first two weeks, in vivo biomechanical measurements showed similar values regardless of the treatment used. However, 2 weeks after surgery, the rhBMP-2 group had a substantial increase in stiffness, which was supported by the imaging modalities. Although the rhGDF-5 group showed comparable mechanical properties at 6 weeks as the rhBMP-2 group, the temporal development of regenerating tissues appeared different with rhGDF-5, resulting in a smaller callus and delayed tissue mineralisation. Moreover, histology showed the presence of cartilage in the rhGDF-5 group whereas the rhBMP-2 group had no cartilaginous tissue. Therefore, this study shows that rhBMP-2 and rhGDF-5 treated defects, under the same conditions, use distinct rates of bone healing as shown by the tissue mechanical properties. Furthermore, results showed that in vivo biomechanical method is capable of detecting differences in healing rate by means of change in callus stiffness due to tissue mineralisation.

Time kinetics of bone defect healing in response to BMP-2 and GDF-5 characterised by in vivo biomechanics

Abstract: This study reports that treatment of osseous defects with different growth factors initiates distinct rates of repair. We developed a new method for monitoring the progression of repair, based upon measuring the in vivo mechanical properties of healing bone. Two different members of the bone morphogenetic protein (BMP) family were chosen to initiate defect healing: BMP-2 to induce osteogenesis, and growth-and-differentiation factor (GDF)-5 to induce chondrogenesis. To evaluate bone healing, BMPs were implanted into stabilised 5 mm bone defects in rat femurs and compared to controls. During the first two weeks, in vivo biomechanical measurements showed similar values regardless of the treatment used. However, 2 weeks after surgery, the rhBMP-2 group had a substantial increase in stiffness, which was supported by the imaging modalities. Although the rhGDF-5 group showed comparable mechanical properties at 6 weeks as the rhBMP-2 group, the temporal development of regenerating tissues appeared different with rhGDF-5, resulting in a smaller callus and delayed tissue mineralisation. Moreover, histology showed the presence of cartilage in the rhGDF-5 group whereas the rhBMP-2 group had no cartilaginous tissue. Therefore, this study shows that rhBMP-2 and rhGDF-5 treated defects, under the same conditions, use distinct rates of bone healing as shown by the tissue mechanical properties. Furthermore, results showed that in vivo biomechanical method is capable of detecting differences in healing rate by means of change in callus stiffness due to tissue mineralisation.

Immunologically restricted patients exhibit a pronounced inflammation and inadequate response to hypoxia in fracture hematomas

Abstract: For patients who are known to have an impaired immune system, bone healing is often impaired. Therefore, it has been suggested that an effectively functioning immune system will have an influence on the quality of bone healing. Here, we demonstrate that cells within the fracture hematoma of immunologically restricted patients (1) exhibit a disturbed osteogenic differentiation (normal SPP1 but diminished RUNX2 expression), (2) show a strong inflammatory reaction (high IL8 and CXCR4), and (3) react on local hypoxia (high expression of HIF1A) but with inadequate target gene responses (diminished LDHA and PGK1 expression). Thus, it is already within the early inflammatory phase of fracture healing that the local gene expression in fracture hematomas of immunologically restricted patients points toward a critical regeneration.

Poorly Ordered Bone as an Endogenous Scaffold for the Deposition of Highly Oriented Lamellar Tissue in Rapidly Growing Ovine Bone

Abstract: The mechanical properties of bone are known to depend on its structure at all length scales. In large animals, such as sheep, cortical bone grows very quickly and it is known that this occurs in 2 sta

Osteogenic Predifferentiation of Human Bone Marrow-Derived Stem Cells by Short-Term Mechanical Stimulation

Abstract: It is commonly accepted that bone marrow-derived stem cells (BMSCs) have to be expanded in vitro, but a prolonged time in culture decreases their multilineage potential. Mechanical and biological stimuli have been used to improve their osteogenic potential. While long-term stimulation has been shown to improve osteogenic differentiation, it remains to be seen whether short-term stimulation is also sufficient. We investigated the influence of 24 hours' cyclic loading (0.05Hz, 4kPa) on gene expression of human BMSCs in three-dimensional fibrin-DMEM constructs (n=7) in a compression bioreactor using DNA-array technology. Expression of the following genes showed a significant increase after mechanical stimulation: 2.6-fold osteopontin (OPN) and integrin-β1 (ITGB1), 2.2-fold transforming growth factor-β-receptor 1 (TGF-β-R1) and 2.4-fold SMAD5 expression, compared to controls without mechanical stimulation (p<0.05 each). Platelet-derived growth factor-α (PDGF-α ) and annexin-V were also significantly overexpressed, the mechanical stimulation resulting in a 1.8-fold and 1.6-fold expression (p<0.05). Cells were identified as osteoblast precursors with a high proliferative capacity. Given the identical in-vitro environment for both groups, the increase in gene expression has been interpreted as a direct influence of cyclic mechanical stimulation on osteogenic differentiation. It may be postulated that short-term mechanical stimulation results in an improved osseous integration of tissue engineered grafts in bone defect healing.

Influence of gender and fixation stability on bone defect healing in middle-aged rats: a pilot study.

Abstract: Gender and stability of fixation independently influence bone regeneration but their combined effects are unclear. In a pilot study we determined the combined influence of gender and fixation stability on the callus of middle-aged rats regarding (1) biomechanical properties; (2) bridging over time; (3) callus formation; and (4) callus size, geometry, mineralization, and microstructure. We osteotomized the left femur of 32 Sprague-Dawley rats (12 months old). Femurs were externally fixed with a gap of 1.5 mm in four groups of eight animals each: female semirigid, male semirigid, female rigid, and male rigid. Qualitative and quantitative in vivo radiographic analyses were performed twice weekly. Six weeks postoperatively, harvested femora were evaluated using micro-CT and biomechanical testing. Torsional stiffness and maximum torque at failure were higher in male and in semirigidly fixed fractures. Radiographic analysis revealed earlier bridging and callus formation in both male groups. Micro-CT analysis showed a larger callus size, altered geometry, and microstructure in males and semirigidly fixed animals, whereas mineralization was similar in all animals. Our data suggest female gender represents an independent risk factor for bone healing in middle-aged rats. Although healing in females was delayed compared with males, they exhibited a similar response (superior callus properties) to a more semirigid fixation. While female gender appears to reflect a risk for impaired bone healing in middle-aged female rats, clinical studies would be required to confirm the finding in humans.

Influence of prosthesis design and implantation technique on implant stresses after cementless revision THR

Abstract: Femoral offset influences the forces at the hip and the implant stresses after revision THR. For extended bone defects, these forces may cause considerable bending moments within the implant, possibly leading to implant failure. This study investigates the influences of femoral anteversion and offset on stresses in the Wagner SL revision stem implant under varying extents of bone defect conditions. Wagner SL revision stems with standard (34 mm) and increased offset (44 mm) were virtually implanted in a model femur with bone defects of variable extent (Paprosky I to IIIb). Variations in surgical technique were simulated by implanting the stems each at 4° or 14° of anteversion. Muscle and joint contact forces were applied to the reconstruction and implant stresses were determined using finite element analyses. Whilst increasing the implant's offset by 10 mm led to increased implant stresses (16.7% in peak tensile stresses), altering anteversion played a lesser role (5%). Generally, larger stresses were observed with reduced bone support: implant stresses increased by as much as 59% for a type IIIb defect. With increased offset, the maximum tensile stress was 225 MPa. Although increased stresses were observed within the stem with larger offset and increased anteversion, these findings indicate that restoration of offset, key to restoring joint function, is unlikely to result in excessive implant stresses under routine activities if appropriate fixation can be achieved.

Quantification and significance of fluid shear stress field in biaxial cell stretching device.

Abstract: A widely used commercially available system for the investigation of mechanosensitivity applies a biaxial strain field to cells cultured on a compliant silicone substrate membrane stretched over a central post. As well as intended substrate strain, this device also provides a fluid flow environment for the cultured cells. In order to interpret the relevance of experiments using this device to the in vivo and clinical situation, it is essential to characterise both substrate and fluid environments. While previous work has detailed the substrate strain, the fluid shear stresses, to which bone cells are known to be sensitive, are unknown. Therefore, a fluid structure interaction computational fluid dynamics model was constructed, incorporating a finite element technique capable of capturing the contact between the post and the silicone substrate membrane, to the underside of which the pump control pressure was applied. Flow verification experiments using 10-μm-diameter fluorescent microspheres were carried out. Fluid shear stress increased approximately linearly with radius along the on-post substrate membrane, with peak values located close to the post edge. Changes in stimulation frequency and culture medium viscosity effected proportional changes in the magnitude of the fluid shear stress (peak fluid shear stresses varied in the range 0.09–3.5 Pa), with minor effects on temporal and spatial distribution. Good agreement was obtained between predicted and measured radial flow patterns. These results suggest a reinterpretation of previous data obtained using this device to include the potential for a strong role of fluid shear stress in mechanosensitivity.

Combined In Vivo/In Silico Study of Mechanobiological Mechanisms During Endochondral Ossification in Bone Healing

Abstract: Mechanobiological theories have been introduced to illustrate the interaction between biology and the local mechanical environment during bone healing. Although several theories have been proposed, a quantitative validation using histomorphometric data is still missing. In this study, in vivo histological data based on an ovine animal experiment was quantified and used to validate bone healing simulations focussing on the endochondral ossification process. The bone formation at different callus regions (periosteal and endosteal bone at the medial and lateral side) was analyzed from in vivo data and quantitatively compared with in silico results. A histomorphometric difference was found in medial and lateral hard callus formation 3 weeks after osteotomy. However, the same amount of new bone was formed on both sides between week 3 and 6. Using a parametric approach, distinct ranges for mechanical strain levels regulating tissue formation were found, for which the in silico prediction agrees with the in vivo endochondral ossification both in pattern and quantity. According to this finding, a strain range of 1 to 8% seems to be conducive for cartilage formation while bone formation may be facilitated by strains up to 4%. This study demonstrates the potential of a thorough validation of in silico results for gaining a better understanding of mechanobiological mechanisms during bone healing.

The challenges of modern interdisciplinary medical research

Abstract: The increasing complexity of medical science poses significant challenges to medical education, leading to a growing gap between medical researchers and treating practitioners.

Skeletal muscle regeneration using mesenchymal system cells

Abstract: The present invention relates to a therapeutic substance and/or medicament and methods relating to the use of said substance and/or medicament for skeletal muscle regeneration using mesenchymal stem cells (MSCs) which can be applied directly or shortly after muscle damage or injury.

Biomechanik des Kniegelenks

Abstract: Um die Zeitraume zu minimieren, die nach einem endoprothetischen Gelenkersatz zur Regeneration des Patienten notig sind, und eine moglichst schnelle und weitestgehende Wiederherstellung der Funktion wahrend der Rehabilitation zu ermoglichen, ist ein gewisses Verstandnis der mechanischen Bedingungen notig. Wissen um die mechanischen Bedingungen ist insbesondere deshalb von Bedeutung, weil zum einen die Endoprothese die makroskopischen Bedingungen fur das Gelenk grundlegend andert. Zum anderen bestimmt das chirurgische Vorgehen – beim Kniegelenk besonders die Balance und Aktivitat der Weichteile – die mechanischen Rahmenbedingungen, unter denen die knocherne Integration des Implantats stattfindet. Somit sind die biomechanischen Bedingungen nicht nur fur die Funktion, sondern auch fur die Dauerhaftigkeit des Gelenkersatzes in dem sich mit dem Alter andernden biologischen Umfeld entscheidend.

The mechanical heterogeneity of the hard callus influences local tissue strains during bone healing

Abstract: During secondary fracture healing, various tissue types including new bone are formed. The local mechanical strains play an important role in tissue proliferation and differentiation. To further our mechanobiological understanding of fracture healing, a precise assessment of local strains is mandatory. Until now, static analyses using Finite Elements (FE) have assumed homogenous material properties. With the recent quantification of both the spatial tissue patterns (Vetter et al., 2010) and the development of elastic modulus of newly formed bone during healing (Manjubala et al., 2009 ), it is now possible to incorporate this heterogeneity. Therefore, the aim of this study is to investigate the effect of this heterogeneity on the strain patterns at six successive healing stages. The input data of the present work stemmed from a comprehensive cross-sectional study of sheep with a tibial osteotomy (Epari et al., 2006 ). In our FE model, each element containing bone was described by a bulk elastic modulus, which depended on both the local area fraction and the local elastic modulus of the bone material. The obtained strains were compared with the results of hypothetical FE models assuming homogeneous material properties. The differences in the spatial distributions of the strains between the heterogeneous and homogeneous FE models were interpreted using a current mechanobiological theory ( Isakson et al., 2006 ). This interpretation showed that considering the heterogeneity of the hard callus is most important at the intermediate stages of healing, when cartilage transforms to bone via endochondral ossification.

The pin-bone interface in external fixator: a standardized analysis in a sheep osteotomy model.

Abstract: Objectives: The hypothesis of the study was that the incidence of pin loosening and pin infection would increase, whereas the general stability of the pin―bone interface would decrease with ongoing implantation time. The aim of this study was to analyze the biologic reactions of the bone tissue adjacent to the pin to determine the relationship among the osseous anchorage of pins, the incidence of infections, and the histologic appearance. Methods: Three groups of sheep received a tibial osteotomy stabilized by external fixators. The pin―bone interface was analyzed biomechanically, radiologically, microbiologically, and histologically after 3, 6, and 9 weeks. Results/Conclusions: Contrary to common opinion, pin anchorage was not altered biomechanically throughout the 9 weeks of the study. This effect might be attributed to an increasing remodeling found in the callus and cortex around the pins and was likely assisted by a strict pin care routine and a low infection rate.