safranine-t and Fractures--Bone

Assessment of the early stages of fracture healing via X-rays and computed tomography is limited by the low radio-opacity of cartilage. We validated a method of contrast-enhanced computed tomography (CECT) for non-destructive identification of cartilage within a healing fracture callus. Closed, stabilized fractures in femora of C57BL/6 mice were harvested on post-operative day 9.5 and imaged ex vivo with micro-computed tomography (µCT) before and after incubation in a cationic contrast agent that preferentially accumulates in cartilage due to the high concentration of sulfated glycosaminoglycans in the tissue. Co-registration of the pre- and post-incubation images, followed by image subtraction, enabled two- and three-dimensional delineation of mineralized tissue, soft callus, and cartilage. The areas of cartilage and callus identified with CECT were compared to those identified with the gold-standard method of histomorphometry. No difference was found between the areas of cartilage measured by the two methods (p = 0.999). Callus area measured by CECT was smaller than, but strongly predictive of (R(2) = 0.80, p < 0.001), the corresponding histomorphometric measurements. CECT also enabled qualitative identification of mineralized cartilage. These findings indicate that the CECT method provides accurate, quantitative, and non-destructive visualization of the shape and composition of the fracture callus, even during the early stages of repair when little mineralized tissue is present. The non-destructive nature of this method would allow subsequent analyses, such as mechanical testing, to be performed on the callus, thus enabling higher-throughput, comprehensive investigations of bone healing.

Topics: Animals; Bony Callus; Cartilage; Contrast Media; Fracture Healing; Fractures, Bone; Male; Mice; Mice, Inbred C57BL; Phenazines; Tomography, X-Ray Computed; X-Ray Microtomography

In this study, hydrogel scaffolds, based on the polymer oligo(poly(ethylene glycol) fumarate) (OPF), were implanted into osteochondral defects in the rabbit model. Scaffolds consisted of two layers-a bottom, bone forming layer and a top, cartilage forming layer. Three scaffold formulations were implanted to assess how material composition and transforming growth factor-beta1 (TGF-beta1) loading affected osteochondral repair. Critical histological evaluation and scoring of the quantity and quality of tissue in the chondral and subchondral regions of defects was performed at 4 and 14 weeks. At both time points, no evidence of prolonged inflammation was observed, and healthy tissue was seen to infiltrate the defect area. The quality of this tissue improved over time with hyaline cartilage filling the chondral region and a mixture of trabecular and compact bone filling the subchondral region at 14 weeks. A promising degree of Safranin O staining and chondrocyte organization was observed in the newly formed surface tissue, while the underlying subchondral bone was completely integrated with the surrounding bone at 14 weeks. Material composition within the bottom, bone-forming layer did not appear to affect the rate of scaffold degradation or tissue filling. However, no bone upgrowth into the chondral region was observed with any scaffold formulation. TGF-beta1 loading in the top layer of scaffolds appeared to exert some therapeutic affect on tissue quality, but further studies are necessary for scaffold optimization. Yet, the excellent tissue filling and integration resulting from osteochondral implantation of these OPF-based scaffolds demonstrates their potential in cartilage repair strategies.

Topics: Absorbable Implants; Animals; Biocompatible Materials; Bone and Bones; Bone Diseases; Bone Substitutes; Cartilage; Cartilage, Articular; Chondrocytes; Coloring Agents; Disease Models, Animal; Fractures, Bone; Gelatin; Hydrogel, Polyethylene Glycol Dimethacrylate; Hydrogels; Inflammation; Lipid Bilayers; Materials Testing; Osteochondritis; Phenazines; Polyesters; Polyethylene Glycols; Rabbits; Regression Analysis; Time Factors; Tissue Engineering; Transforming Growth Factor beta; Transforming Growth Factor beta1; Wound Healing