Insights into mesenchymal stem cell aging: involvement of antioxidant defense and actin cytoskeleton.

Progenitor cells such as mesenchymal stem cells (MSCs) have elicited great hopes for therapeutic augmentation of physiological regeneration processes, e.g., for bone fracture healing. However, regeneration potential decreases with age, which raises questions about the efficiency of autologous approaches in elderly patients. To elucidate the mechanisms and cellular consequences of aging, the functional and proteomic changes in MSCs derived from young and old Sprague-Dawley rats were studied concurrently. We demonstrate not only that MSC concentration in bone marrow declines with age but also that their function is altered, especially their migratory capacity and susceptibility toward senescence. High-resolution two-dimensional electrophoresis of the MSC proteome, under conditions of in vitro self-renewal as well as osteogenic stimulation, identified several age-dependent proteins, including members of the calponin protein family as well as galectin-3. Functional annotation clustering revealed that age-affected molecular functions are associated with cytoskeleton organization and antioxidant defense. These proteome screening results are supported by lower actin turnover and diminished antioxidant power in aged MSCs, respectively. Thus, we postulate two main reasons for the compromised cellular function of aged MSCs: (a) declined responsiveness to biological and mechanical signals due to a less dynamic actin cytoskeleton and (b) increased oxidative stress exposure favoring macromolecular damage and senescence. These results, along with the observed similar differentiation potentials, imply that MSC-based therapeutic approaches for the elderly should focus on attracting the cells to the site of injury and oxidative stress protection, rather than merely stimulating differentiation.

Locally applied osteogenic predifferentiated progenitor cells are more effective than undifferentiated mesenchymal stem cells in the treatment of delayed bone healing.

Mesenchymal stem cells (MSCs) and osteogenic predifferentiated cells (OPCs) have been shown to promote healing of critical-sized bone defects. This study investigated the regenerative capacity of autologous MSCs versus OPCs after local injection into a compromised bone healing situation. We hypothesized that treatment with MSCs and OPCs would enhance the healing and that the MSCs would be more effective due to their lower differentiation and higher proliferative competence. The femur of rats was osteotomized and stabilized with an external fixator. Except for the control group (C group), in all animals a delayed healing was induced by cautering the periosteum and removing the bone marrow. Two days postsurgery, autologous MSCs (MSC group), OPCs (OPC group), or cell-free medium (Sham group) was percutaneously injected into the osteotomy gap. The C group received no treatment. Bone healing was evaluated radiologically, biomechanically, and histologically. After 8 weeks, the C group showed complete bony bridging, while a delayed healing was detected in the Sham group. All outcome measures showed better healing of the OPC group compared to the Sham group. Contrary to our expectations, there were no significant differences in outcome measures between the MSC group and the Sham group. The percutaneous injection of OPCs could become a minimally invasive treatment option for delayed or nonunions.

A new animal model for bone atrophic nonunion: fixation by external fixator.

A new small animal model of bone atrophic nonunion was established for investigating the process of bone regeneration by performing cauterization of the periosteum, removal of the local bone marrow, and stabilization with external fixation. The model allows the creation of an atrophic nonunion without the need for a critical size defect. Furthermore, it provides reproducible, well-defined mechanical conditions and minimized physical interference of the implant with the biological processes in the healing zone. Eighty adult Sprague-Dawley rats received an osteotomy of the left femur, stabilized with an external fixator. In half of the animals, the periosteum proximal and distal to the osteotomy was destroyed by cauterization and the adjacent bone marrow was removed (nonunion group). At 2 and 8 weeks after surgery, radiological, biomechanical, histological, and histomorphometrical analyses showed a typical physiological healing in the control group, while the nonunion group was characterized by resorption of the bone ends with some callus formation distant to the osteotomy. At both time points, the callus was composed of significantly less bone and significantly more connective tissue (p < 0.001). In addition, the torsional strength of the osteotomized femur was significantly less in the nonunion group than in the control group, which was comparable to that of the intact femur (p < 0.001). In conclusion, the present model allows the induction of an atrophic nonunion without the need of a critical size defect. It is reproducible, provides standardized biomechanical conditions, and allows minimized interaction of the implant with the healing zone.

Influence of age and mechanical stability on bone defect healing: age reverses mechanical effects.

Non-unions and delayed healing are still prevalent complications in fracture and bone defect healing. Both mechanical stability and age are known to influence this process. However, it remains unclear which factor dominates and how they interact. Within this study, we sought a link between both factors. In 36 female Sprague-Dawley rats, the left femur was osteotomized, distracted to an osteotomy gap of 1.5 mm and externally fixated. Variation of age (12 vs. 52 weeks - biologically challenging) and fixator stiffness (mechanically challenging) resulted in 4 groups (each 9 animals): YS: young semi-rigid, OS: old semi-rigid, YR: young rigid and OR: old rigid. Qualitative and quantitative radiographical analyses were performed at weeks 2, 4 and 6 after surgery. Six weeks post-op, rats were sacrificed and femora were harvested for biomechanical testing (torsional stiffness (TS) and maximum torque at failure (MTF)). Six weeks after surgery, TS showed a significant interaction between age and fixation stiffness (p<0.0001). TS in YR was significantly higher than that in the other groups (YS: p<0.001; OR: p<0.001; OS: p<0.001). Additionally, YS showed a significantly higher TS compared to the OS (p=0.006) and OR (p=0.046). Testing of MTF showed a significant interaction of both variables (p=0.0002) and led to significant differences between OR and YS (p<0.001), OS (p=0.046) and YR (p<0.001). The YR showed a higher MTF compared to YS (p=0.012) and OS (p=0.001), whereas OR's MTF was inferior compared to OS. At 2-week follow-up, YR (p=0.006), and at 6-week follow-up, YS and YR (p=0.032) showed significantly higher radiographic scores. At 2-week follow-up, YS's callus was larger than that of the old groups (OS: p=0.025; OR: p=0.003). In YR a significantly smaller callus was observed compared to YS at time points 4 and 6 weeks (p=0.002 for both) and compared to OS at 6-week follow-up (p=0.03). The effect of age seems to invert the effect of mechanical properties of the callus, which was not correlated to callus size. Optimization of mechanics alone seems to be not sufficient. The underlying mechanisms and causes of the age-related influences and their clinical counterparts need to be further investigated.

An easily reproducible and biomechanically standardized model to investigate bone healing in rats, using external fixation.

Abstract We have established a new small animal model to investigate the process of bone regeneration. A total of 42 male Sprague-Dawley rats received an osteotomy of the left femur, stabilized with a custom-made external fixator. The fixation method was chosen to create an easily reproducible, biomechanically well-defined model with minimized interference of the implant with the healing zone. At 14 or 56 days post-operation, the animals were sacrificed and examined biomechanically, histologically and radiologically. Radiologically, the femurs of all animals were anatomically positioned directly post-operation and remained in that position throughout the examination period. At 14 days post-operation, a typical periosteal callus formation could be observed both histologically and radiologically. At 56 days post-operation, the osteotomy was almost completely bridged by periosteal callus and the biomechanical competence of the bones was fully restored. Relative to the intact contralateral femur, the torsional stiffness median was 130.3% (interquartile range 118.9-157.7%) and the maximum torsional failure moment median was 135.6% (interquartile range 69.5-208.7%). As this model provides standardized conditions, it is suitable for a wide range of investigations and is particularly valuable for investigations of locally applied therapies, such as osteoconductive materials or osteoinductive factors.