The specialist in regeneration-the Axolotl-a suitable model to study bone healing?

While the axolotl's ability to completely regenerate amputated limbs is well known and studied, the mechanism of axolotl bone fracture healing remains poorly understood. One reason might be the lack of a standardized fracture fixation in axolotl. We present a surgical technique to stabilize the osteotomized axolotl femur with a fixator plate and compare it to a non-stabilized osteotomy and to limb amputation. The healing outcome was evaluated 3 weeks, 3, 6 and 9 months post-surgery by microcomputer tomography, histology and immunohistochemistry. Plate-fixated femurs regained bone integrity more efficiently in comparison to the non-fixated osteotomized bone, where larger callus formed, possibly to compensate for the bone fragment misalignment. The healing of a non-critical osteotomy in axolotl was incomplete after 9 months, while amputated limbs efficiently restored bone length and structure. In axolotl amputated limbs, plate-fixated and non-fixated fractures, we observed accumulation of PCNA+ proliferating cells at 3 weeks post-injury similar to mouse. Additionally, as in mouse, SOX9-expressing cells appeared in the early phase of fracture healing and amputated limb regeneration in axolotl, preceding cartilage formation. This implicates endochondral ossification to be the probable mechanism of bone healing in axolotls. Altogether, the surgery with a standardized fixation technique demonstrated here allows for controlled axolotl bone healing experiments, facilitating their comparison to mammals (mice).

A biomaterial with a channel-like pore architecture induces endochondral healing of bone defects.

Biomaterials developed to treat bone defects have classically focused on bone healing via direct, intramembranous ossification. In contrast, most bones in our body develop from a cartilage template via a second pathway called endochondral ossification. The unsolved clinical challenge to regenerate large bone defects has brought endochondral ossification into discussion as an alternative approach for bone healing. However, a biomaterial strategy for the regeneration of large bone defects via endochondral ossification is missing. Here we report on a biomaterial with a channel-like pore architecture to control cell recruitment and tissue patterning in the early phase of healing. In consequence of extracellular matrix alignment, CD146+ progenitor cell accumulation and restrained vascularization, a highly organized endochondral ossification process is induced in rats. Our findings demonstrate that a pure biomaterial approach has the potential to recapitulate a developmental bone growth process for bone healing. This might motivate future strategies for biomaterial-based tissue regeneration.

In serum veritas-in serum sanitas? Cell non-autonomous aging compromises differentiation and survival of mesenchymal stromal cells via the oxidative stress pathway.

Even tissues capable of complete regeneration, such as bone, show an age-related reduction in their healing capacity. Here, we hypothesized that this decline is primarily due to cell non-autonomous (extrinsic) aging mediated by the systemic environment. We demonstrate that culture of mesenchymal stromal cells (MSCs) in serum from aged Sprague-Dawley rats negatively affects their survival and differentiation ability. Proteome analysis and further cellular investigations strongly suggest that serum from aged animals not only changes expression of proteins related to mitochondria, unfolded protein binding or involved in stress responses, it also significantly enhances intracellular reactive oxygen species production and leads to the accumulation of oxidatively damaged proteins. Conversely, reduction of oxidative stress levels in vitro markedly improved MSC function. These results were validated in an in vivo model of compromised bone healing, which demonstrated significant increase regeneration in aged animals following oral antioxidant administration. These observations indicate the high impact of extrinsic aging on cellular functions and the process of endogenous (bone) regeneration. Thus, addressing the cell environment by, for example, systemic antioxidant treatment is a promising approach to enhance tissue regeneration and to regain cellular function especially in elderly patients.

An experimental setup to evaluate innovative therapy options for the enhancement of bone healing using BMP as a benchmark--a pilot study.

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 fixation 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 artificial 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 ISH-group. 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.

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

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.