Robert Blakytny

Review: The Diabetic Bone: A Cellular and Molecular Perspective

With the increasing worldwide prevalence of diabetes the resulting complications, their consequences and treatment will lead to a greater social and financial burden on society. One of the many organs to be affected is bone. Loss of bone is observed in type 1 diabetes, in extreme cases mirroring osteoporosis, thus a greater risk of fracture. In the case of type 2 diabetes, both a loss and an increase of bone has been observed, although in both cases the quality of the bone overall was poorer, again leading to a greater risk of fracture. Once a fracture has occurred, healing is delayed in diabetes, including nonunion. The reasons leading to such changes in the state of the bone and fracture healing in diabetes is under investigation, including at the cellular and the molecular levels. In comparison with our knowledge of events in normal bone homeostasis and fracture healing, that for diabetes is much more limited, particularly in patients. However, progress is being made, especially with the use of animal models for both diabetes types. Identifying the molecular and cellular changes in the bone in diabetes and understanding how they arise will allow for targeted intervention to improve diabetic bone, thus helping to counter conditions such as Charcot foot as well as preventing fracture and accelerating healing when a fracture does occur.

Fracture healing in mice under controlled rigid and flexible conditions using an adjustable external fixator.

Mice are increasingly used to investigate mechanobiology in fracture healing. The need exists for standardized models allowing for adjustment of the mechanical conditions in the fracture gap. We introduced such a model using rigid and flexible external fixators with considerably different stiffness (axial stiffnesses of 18.1 and 0.82 N/mm, respectively). Both fixators were used to stabilize a 0.5 mm osteotomy gap in the femur of C57BL/6 mice (each n = 8). Three‐point bending tests, µCT, and histomorphometry demonstrated a different healing pattern after 21 days. Both fixations induced callus formation with a mixture of intramembranous and enchondral ossification. Under flexible conditions, the bending stiffness of the callus was significantly reduced, and a larger but qualitatively inferior callus with a significantly lower fraction of bone but a higher fraction of cartilage and soft tissue was formed. Monitoring of the animal movement and the ground reaction forces demonstrated physiological loading with no significant differences between the groups, suggesting that the differences in healing were not based on a different loading behavior. In summary, flexible external fracture fixation of the mouse femur led to delayed fracture healing in comparison to a more rigid situation.

Early dynamization by reduced fixation stiffness does not improve fracture healing in a rat femoral osteotomy model

Dynamization of fracture fixation is used clinically to improve the bone healing process. However, the effect of early dynamization remains controversial. This study evaluated the effect of early dynamization, by reduced stiffness of fixation on callus stiffness and size after 5 weeks of healing in a rat diaphyseal femoral osteotomy. An external unilateral fixator allowed either a rigid (R‐group; n = 8) or a flexible (F‐group; n = 8) fixation. The dynamized group (D‐group: n = 8) had a rigid fixation for 1 week, and then a flexible fixation for the remaining 4 weeks. The pre‐ and postoperative activity of the rats was measured. After 5 weeks, the rats were sacrificed, and healing was evaluated by biomechanical and densitometric methods. The R‐group had a higher activity more closely approaching preoperative levels, compared to the D‐group throughout all time points measured. This difference was significant after 14 days and 21 days. The flexural rigidity of the R‐group was 82% (tested in the anterior‐posterior direction; p = 0.01) and 93% (tested in the medial‐lateral direction; p = 0.002) greater than the flexural rigidity of the D‐group. The rigid fixation led to a stiffer callus with a smaller callus volume, but better mineralized tissue in the whole callus and at the level of the osteotomy gap than the flexible or the dynamized fixation. Early dynamization did not improve healing compared to rigid or flexible fixation in a rat femoral osteotomy model.

Altered molecular mechanisms of diabetic foot ulcers.

The continuously increasing worldwide prevalence of diabetes will be accompanied by a greater incidence of diabetic foot ulcer, a complication in which many of the morphological processes involved in normal wound healing are disrupted. The highly complex and integrated process of wound healing is regulated by a large array of molecular factors. These often have overlapping functions, ensuring a certain degree of tolerance through redundancy. In diabetes, changes to the expression of a large number of molecular factors have been observed, overwhelming this inbuilt redundancy. This results in delayed healing or incomplete healing as in ulceration. Understanding the relationship between altered levels of molecular factors and the inhibited healing process in such ulcers will permit the development of targeted treatments aimed to greatly improve the quality of life of patients, at the same time helping to reduce the huge costs associated with treating this diabetic condition and its long-term consequences. This short review examines how changes in the expression of molecular factors are related to altered morphology in diabetic foot ulceration and very briefly considers treatment strategies at molecular level.

The Anaphylatoxin Receptor C5aR Is Present During Fracture Healing in Rats and Mediates Osteoblast Migration In Vitro

BACKGROUND There is evidence that complement components regulate cytokine production in osteoblastic cells, induce cell migration in mesenchymal stem cells, and play a regulatory role in normal enchondral bone formation. We proved the hypothesis that complement might be involved in bone healing after fracture. METHODS We investigated the expression of the key anaphylatoxin receptor C5aR during fracture healing in rats by immunostaining after 1, 3, 7, 14, and 28 days. C5aR expression was additionally analyzed in human mesenchymal stem cells (hMSC) during osteogenic differentiation, in human primary osteoblasts, and osteoclasts by reverse transcriptase polymerase chain reaction and immunostaining. Receptor functionality was proven by the migratory response of cells to C5a in a Boyden chamber. RESULTS C5aR was expressed in a distinct spatial and temporal pattern in the fracture callus by differentiated osteoblast, chondroblast-like cells in cartilaginous regions, and osteoclasts. In vitro C5aR was expressed by osteoblasts, osteoclasts, and hMSC undergoing osteogenic differentiation. C5aR was barely expressed by undifferentiated hMSC but was significantly induced after osteogenic differentiation. C5aR activation by C5a induced strong chemotactic activity in osteoblasts, and in hMSC, which had undergone osteogenic differentiation, being abolished by a specific C5aR antagonist. In hMSC, C5a induced less migration reflecting their low level of C5aR expression. CONCLUSIONS Our in vitro and in vivo results demonstrated the presence of C5aR in bone forming osteoblasts and bone resorbing osteoclasts. It is suggested that C5aR might play a regulatory role in fracture healing in intramembranous and in enchondral ossification, one possible function being the regulation of cell recruitment.

TGF-β–dependent suppressive function of Tregs requires wild-type levels of CD18 in a mouse model of psoriasis

Dysfunctional Tregs have been identified in individuals with psoriasis. However, their role in the pathogenesis of the disease remains unclear. Here we explored the effect of diminished CD18 (beta2 integrin) expression on the function of CD4+CD25+CD127(-) Tregs using the Cd18 hypomorphic (Cd18hypo) PL/J mouse model of psoriasis that closely resembles the human disease. We found that reduced CD18 expression impaired cell-cell contact between Tregs and DCs. This led to dysfunctional Tregs, which both failed to suppress the pathogenic T cells and promoted the onset and severity of the disease. This failure was TGF-beta-dependent, as Tregs derived from Cd18hypo PL/J mice had diminished TGF-beta1 expression. Adoptive transfer of Tregs expressing wild-type levels of CD18 into affected Cd18hypo PL/J mice resulted in a substantial improvement of the psoriasiform skin disease, which did not occur upon coinjection of the cells with TGF-beta-specific neutralizing antibody. Our data indicate a primary dysfunction of Cd18hypo Tregs, allowing subsequent hyperproliferation of pathogenic T cells in the Cd18hypo PL/J mouse model of psoriasis. This study may provide a step forward in our understanding of the unique role of CD18 expression levels in avoiding autoimmunity.

Late dynamization by reduced fixation stiffness enhances fracture healing in a rat femoral osteotomy model.

Objectives: This study evaluated the effect of late dynamization on callus stiffness and size in a rat diaphyseal femoral osteotomy model. It was hypothesized that late dynamization, after bony bridging, would enhance healing. Methods: The external unilateral fixator was dynamized by removal of the inner fixator bar at either 3 weeks (D3 group) or 4 weeks (D4 group) postoperation. After 5 weeks, the rats were euthanized and healing was evaluated by biomechanical and densitometric methods. Published data of control groups, constant rigid (R group), and flexible fixation (F group) were included for comparison. Results: Enhanced healing was observed in both the 3- and 4-week dynamized groups compared with the constant flexible and constant rigid fixation. Late dynamization after both 3 and 4 weeks postoperation led to a stiffer callus (percent flexural rigidity relative to the intact side and elastic modulus) with a smaller callus bone volume compared with the F group. Both late dynamized groups (D3 and D4) had a similar flexural rigidity and bone mineral density as that of the R group. However, the D4 group had a significantly greater elastic modulus and significantly smaller callus bone volume compared with the R group suggesting increased remodeling occurred in the D4 group indicative of more advanced healing. Conclusions: Late dynamization enhanced fracture healing in this animal model compared with a constant rigid or constant flexible fixation. Although results of the small animal study cannot directly be transferred to humans, these results indicate that once bony bridging has occurred, dynamization may accelerate bone remodeling processes.

A 9-Centimorgan Interval of Chromosome 10 Controls the T Cell-Dependent Psoriasiform Skin Disease and Arthritis in a Murine Psoriasis Model

Psoriasis is a complex genetic disease of unresolved pathogenesis with both heritable and environmental factors contributing to onset and severity. In addition to a disfiguring skin inflammation, approximately 10–40% of psoriasis patients suffer from destructive joint involvement. Previously, we reported that the CD18 hypomorphic PL/J mouse carrying a mutation resulting in reduced expression of the common chain of β2 integrins (CD11/CD18) spontaneously develops a skin disease that closely resembles human psoriasis. In contrast, the same mutation on C57BL/6J background did not demonstrate this phenotype. By a genome-wide linkage analysis, two major loci were identified as contributing to the development of psoriasiform dermatitis under the condition of low CD18 expression. Using a congenic approach, we now demonstrate that the introduction of a 9-centimorgan fragment of chromosome 10 derived from the PL/J strain into the disease-resistant CD18 hypomorphic C57BL/6J was promoting the development of psoriasiform skin disease and notably also arthritis. We therefore designated this locus psoriasiform skin disease-associated locus 1 (PSD1). High numbers of CD4+ T cells and TNF-α producing macrophages were detected both in inflamed skin and joints in these congenic mice, with a complete resolution upon TNF-α inhibitor therapy or depletion of CD4+ T cells. For the first time, we have identified a distinct genetic element that contributes to the T cell-dependent development of both psoriasiform skin disease and associated arthritis. This congenic model will be suitable to further investigations of genetic and molecular pathways that cause psoriasiform dermatitis and arthritis, and it may also be relevant for other autoimmune diseases.

Discretization error when using finite element models: Analysis and evaluation of an underestimated problem

Mesh convergence tests are often insufficiently performed in finite element analyses. There are many parameters which may have an effect on the mesh convergence behavior. The aim of this study was to identify the influence of different parameters on the mesh convergence behavior. For this purpose we used a simplified axis-symmetrical model of a single pedicle screw flank with surrounding bone to simulate a pull-out test. In parameter studies, the flank radii and the contact conditions at the bone-screw interface were varied. These parameter studies were carried out using an implicit and explicit solver. Thereby, the convergence criteria and the number of the substeps for the implicit nonlinear iteration process as well as the velocity and the material density for the explicit approach were considered. The mesh convergence behavior was influenced by varying the flank radii and the contact conditions. The implicit calculations led to a reaction force, which converged rapidly to a certain value with increasing mesh density, whereas the maximum von-Mises stress showed substantial convergence problems. The number of substeps and the convergence criteria of the iteration process strongly influenced the implicit solutions. In contrast, the maximum von-Mises stresses resulting from explicit calculations converged to a certain value after only a few refinement steps. Different pull-out velocities substantially affected the mesh convergence behavior, while the material density showed only a negligible influence. The results indicated the need to perform an appropriate mesh convergence test when using finite element methods. We were able to show that different parameters strongly influence the mesh convergence behavior and we demonstrated that convergence tests do not always lead to a satisfactory or acceptable solution.

Identification of Susceptibility Loci for Skin Disease in a Murine Psoriasis Model

Psoriasis is a frequently occurring inflammatory skin disease characterized by thickened erythematous skin that is covered with silvery scales. It is a complex genetic disease with both heritable and environmental factors contributing to onset and severity. The CD18 hypomorphic PL/J mouse reveals reduced expression of the common chain of β2 integrins (CD11/CD18) and spontaneously develops a skin disease that closely resembles human psoriasis. In contrast, CD18 hypomorphic C57BL/6J mice do not demonstrate this phenotype. In this study, we have performed a genome-wide scan to identify loci involved in psoriasiform dermatitis under the condition of low CD18 expression. Backcross analysis of a segregating cross between susceptible CD18 hypomorphic PL/J mice and the resistant CD18 hypomorphic C57BL/6J strain was performed. A genome-wide linkage analysis of 94 phenotypically extreme mice of the backcross was undertaken. Thereafter, a complementary analysis of the regions of interest from the genome-wide screen was done using higher marker density and further mice. We found two loci on chromosome 10 that were significantly linked to the disease and interacted in an additive fashion in its development. In addition, a locus on chromosome 6 that promoted earlier onset of the disease was identified in the most severely affected mice. For the first time, we have identified genetic regions associated with psoriasis in a mouse model resembling human psoriasis. The identification of gene regions associated with psoriasis in this mouse model might contribute to the understanding of genetic causes of psoriasis in patients and pathological mechanisms involved in development of disease.