Fracture healing is delayed in the absence of gasdermin signaling

Amino-terminal fragments from proteolytically cleaved gasdermins (GSDMs) form plasma membrane pores that enable the secretion of interleukin-1β (IL-1β) and IL-18. Excessive GSDM-mediated pore formation can compromise the integrity of the plasma membrane thereby causing the lytic inflammatory cell death, pyroptosis. We found that GSDMD and GSDME were the only GSDMs that were readily expressed in bone microenvironment. Therefore, we tested the hypothesis that GSDMD and GSDME are implicated in fracture healing owing to their role in the obligatory inflammatory response following injury. We found that bone callus volume and biomechanical properties of injured bones were significantly reduced in mice lacking either GSDM compared with wild-type (WT) mice, indicating that fracture healing was compromised in mutant mice. However, compound loss of GSDMD and GSDME did not exacerbate the outcomes, suggesting shared actions of both GSDMs in fracture healing. Mechanistically, bone injury induced IL-1β and IL-18 secretion in vivo, a response that was mimicked in vitro by bone debris and ATP, which function as inflammatory danger signals. Importantly, the secretion of these cytokines was attenuated in conditions of GSDMD deficiency. Finally, deletion of IL-1 receptor reproduced the phenotype of Gsdmd or Gsdme deficient mice, implying that inflammatory responses induced by the GSDM-IL-1 axis promote bone healing after fracture.

Autologous adipose-derived mesenchymal stem cells and hydroxyapatite for bone defect in rabbits

This study aims to evaluate the effect of autologous adipose-derived mesenchymal stem cells (AAD-MSC), with and without synthetic absorbable hydroxyapatite (HAP-91), on the bone regeneration in rabbits. Thirty-four female white New Zealand rabbits were submitted to a 10 mm distal diaphyseal radius ostectomy, divided into 3 experimental groups according to the treatment established. The bone gap was filled with 0.15 ml of a 0.9% saline solution containing two million AAD-MSC (G1), or AAD-MSC associated with HAP-91 (G2). The control group (CG) received only 0.15 ml of the 0.9% saline solution. Radiographs were made post-operatively, and after 15, 30, 45 and 90 days. Fifty percent of the samples were submitted to a histological examination at 45 days and the remaining ones at 90 days post-operatively. Radiographically, the periosteal reaction, bone callus volume and bone bridge quality were superior in G2 (P < 0.05). Histologically, the bone repair was faster and more efficient in G1 at 45 days (P < 0.05). In conclusion, AAD-MSC improved the regeneration on the experimentally induced bone defects in rabbits; however, the use of hydroxyapatite requires caution given the granulomatous reaction produced in the species.

A Novel Capacitive Measurement Device for Longitudinal Monitoring of Bone Fracture Healing

The healing process of surgically-stabilised long bone fractures depends on two main factors: (a) the assessment of implant stability, and (b) the knowledge of bone callus stiffness. Currently, X-rays are the main diagnostic tool used for the assessment of bone fractures. However, they are considered unsafe, and the interpretation of the clinical results is highly subjective, depending on the clinician’s experience. Hence, there is the need for objective, non-invasive and repeatable methods to allow a longitudinal assessment of implant stability and bone callus stiffness. In this work, we propose a compact and scalable system, based on capacitive sensor technology, able to measure, quantitatively, the relative pins displacements in bone fractures treated with external fixators. The measurement device proved to be easily integrable with the external fixator pins. Smart arrangements of the sensor units were exploited to discriminate relative movements of the external pins in the 3D space with a resolution of 0.5 mm and 0.5°. The proposed capacitive technology was able to detect all of the expected movements of the external pins in the 3D space, providing information on implant stability and bone callus stiffness.

Histological Analysis of Bone Callus in Delayed Union Model Fracture Healing Stimulated with Pulsed Electromagnetic Fields (PEMF)

Delayed union and nonunion fractures are clinical challenges for orthopedic surgeons. The development of fracture complications, such as delayed union and nonunion fractures, is still difficult to predict. Various methods are being investigated to improve fracture healing and prevent complications in patients. There are various methods to promote fracture healing, broadly divided into biological, chemical, and physical methods. One of the most widely used physical methods to promote fracture healing is the pulsed electromagnetic field (PEMF). This study aimed to evaluate the healing process of delayed union fracture after being stimulated by PEMF. Twenty-four rats were randomly divided into two groups: the control group (n = 12) and the PEMF group (n = 12). Delayed union fracture was performed on the left femur of all rats. Subsequently, the PEMF group was given PEMF stimulus with a magnetic field intensity of 1.6 mT and a frequency of 50 Hz for 4 hours/day and 7 days/week. The fracture healing process was evaluated on days 5, 10, 18, and 28 based on the bone callus histology using safranin O fast green (SOFG) staining. The results of the histological analysis showed that bone cartilage was higher in the PEMF group than in the control group throughout the observation period. In addition, the PEMF group had less fibrous tissue at the beginning of the healing. This finding indicates PEMF stimulation has an effect on inducing osteogenesis on fracture healing and reducing the risk of delayed union.

Reparative histogenesis of bone tissue in femoral fractures in rats using biocomposite material along with immunocorrection

The paper studies the effect of the RVI biocomposite material belonging to the group of osteoplastic biocomposite materials, the RV-2 immunomodulator – a synthetic dipeptide inducing an immunocorrective effect, and combinations of these drugs on the reparative histogenesis of bone tissue in femoral fractures in rats. It was found that the remodeling of the primary bone callus into the secondary one in the fracture of the studied animals was of a diverse nature. This process was the most pronounced in the group where the components were used in complex, i.e. the bone defect was filled with RVI during the surgery, as well as RV-2 was injected intramuscularly to rats at a dose of 10 mcg per 1 kg of live weight for five days, starting immediately after the surgery. Well-formed coarse-fibrous connective tissue callus was recorded in animals of this group. The connective tissue was stained more intensely which indicates a denser arrangement of fibers in the callus. Focal cartilage tissue spanning bone fragments was observed within the callus. At the periphery of the site the cartilaginous callus was subjected to endochondral ossification with replacement by coarse-fibrous trabeculae with elements of lamellar bone tissue having haversian canals in the center. The inter-girdle spaces were filled with elements of the myeloid bone marrow in the forming bone tissue. Markedly proliferated osteoblasts were visible in the cambial layer of the periosteum. The bone tissue ratio increased up to (60.21 ± 2.62)%, which significantly exceeded the same indicator in the control group and in all experimental groups. The low content of connective tissue and the high ratio of bone tissue indicated more active osteogenesis processes and reparative regeneration in comparison with other groups.

Axial Micromotion Locking Plate Construct Can Promote Faster and Stronger Bone Healing in an Ovine Osteotomy Model

Fixing bone fractures with controlled axial interfragmentary micromotion improves bone healing; however, the optimal type of implant construct for this purpose is still lacking. The present study describes a novel axial micromotion locking plate (AMLP) construct that allows axial interfragmentary micromotion of 0.3 or 0.6 mm. We investigated whether the AMLP constructs enhance bone healing compared to an ordinary locking plate (LP) using an ovine osteotomy model. The stiffness of the constructs was tested under axial loading. We created a 3-mm osteotomy in the left hind leg tibia of sheep that was then stabilized with a 0.3- or 0.6-mm AMLP or LP construct (n = 6/group). Bone healing was monitored weekly by X-ray radiography starting from week 3 after surgery. At week 9, the specimens were collected and evaluated by computed tomography and torsional testing. We found that the AMLPs had a lower stiffness than the LP; in particular, the stiffness of the 0.6-mm AMLP construct was 86 and 41% lower than that of the LP construct for axial loads <200 and >200 N, respectively. In the in vivo experiments, tibial osteotomies treated with the 0.6-mm AMLP construct showed the earliest maximum callus formation (week 5) and the highest volume of bone callus (9.395 ± 1.561 cm3 at week 9). Specimens from this group also withstood a 27% greater torque until failure than those from the LP group (P = 0.0386), with 53% more energy required to induce failure (P = 0.0474). These results demonstrate that AMLP constructs promote faster and stronger bone healing than an overly rigid LP construct. Moreover, better bone healing was achieved with an axial micromotion of 0.6 mm as compared to 0.3 mm.

Variable fixation promotes callus formation: an experimental study on transverse tibial osteotomies stabilized with locking plates

Background A new locking screw technology, named variable fixation, has been developed aiming at promoting bone callus formation providing initial rigid fixation followed by progressive fracture gap dynamisation. In this study, we compared bone callus formation in osteotomies stabilized with standard locking fixation against that of osteotomies stabilized with variable fixation in an established tibia ovine model. Methods A 3 mm tibial transverse osteotomy gap was stabilized in three groups of six female sheep each with a locking plate and either 1) standard fixation in both segments (group LS) or 2) variable fixation in the proximal and standard fixation in the distal bone segment (group VFLS3) or 3) variable fixation in both segments (group VFLS6). The implantation site and fracture healing were compared between groups by means of radiologic, micro tomographic, biomechanical, and histological investigations. Results Compared to LS callus, VFLS3 callus was 40% larger and about 3% denser, while VFLS6 callus was 93% larger and its density about 7.2% lower. VFLS3 showed 65% and VFLS6 163% larger amount of callus at the cis-cortex. There wasn’t a significant difference in the amount of callus at the cis and trans-cortex in groups featuring variable fixation only. Investigated biomechanical variables were not significantly different among groups and histology showed comparable good healing in all groups. Tissues adjacent to the implants did not show any alteration of the normal structure in all groups. Conclusions Variable fixation promoted the formation of a larger amount of bone callus, equally distributed at the cis and trans cortices. The histological and biomechanical properties of the variable fixation callus were equivalent to those of the standard fixation callus. The magnitude of variable fixation had a biological effect on the formation of bone callus. At the implantation site, the usage of variable fixation did not raise additional concerns with respect to standard fixation. The formation of a larger amount of mature callus suggests that fractures treated with variable fixation might have a higher probability to bridge the fracture gap. The conditions where its usage can be most beneficial for patients needs to be clinically defined.