Perilipin-1 immunostaining improves semi-automated digital quantitation of bone marrow adipocytes in histological bone sections.

Bone marrow adipocytes (BMAds) are not just passive fillers inside the bone marrow compartment but respond to various metabolic changes. Assessment of those responses requires evaluation of the number of BMAds and their morphology for which laborious and error-prone manual histological analysis remains the most widely used method. Here, we report an alternative image analysis strategy to semi-automatically quantitate and analyse the morphology of BMAds in histological bone sections. Decalcified, formalin-fixed paraffin-embedded histological sections of long bones of Sprague-Dawley rats were stained with either haematoxylin and eosin (HE) or by immunofluorescent staining for adipocyte-specific protein perilipin-1 (PLIN1). ImageJ-based commands were constructed to detect BMAds sized 200 µm2 or larger from standardized 1 mm2 analysis regions by either classifying the background colour (HE) or the positive and circular PLIN1 fluorescent signal. Semi-automated quantitation strongly correlated with independent, single-blinded manual counts regardless of the staining method (HE-based: r=0.85, p<0.001; PLIN1 based: r=0.95, p<0.001). The detection error was higher in HE-stained sections than in PLIN1-stained sections (14% versus 5%, respectively; p<0.001), which was due to false-positive detections of unstained adipocyte-like circular structures. In our dataset, the total adiposity area from standardised ROIs in PLIN-1-stained sections correlated with that in whole-bone sections (r=0.60, p=0.02).

Glucose transporters GLUT1, GLUT3, and GLUT4 have different effects on osteoblast proliferation and metabolism.

Bone is an active tissue that undergoes constant remodeling. Bone formation requires energy and one of the energy sources of bone-forming osteoblasts is glucose, which is transported inside the cells via glucose transporters. However, the role of class I glucose transporters in the differentiation and metabolism of osteoblasts and their precursors, bone marrow mesenchymal stromal cells (BMSCs) remains inconclusive. Our aim was to characterize the expression and contribution of main class I glucose transporters, GLUT1, GLUT3, and GLUT4, during osteoblast proliferation and differentiation. To investigate the role of each GLUT, we downregulated GLUTs with siRNA technology in primary rat BMSCs. Live-cell imaging and RNA-seq analysis was used to evaluate downstream pathways in silenced osteoblasts. Glucose transporters GLUT1, GLUT3, and GLUT4 had distinct expression patterns in osteoblasts. GLUT1 was abundant in BMSCs, but rapidly and significantly downregulated during osteoblast differentiation by up to 80% (p < 0.001). Similar downregulation was observed for GLUT4 (p < 0.001). In contrast, expression levels of GLUT3 remained stable during differentiation. Osteoblasts lacked GLUT2. Silencing of GLUT4 resulted in a significant decrease in proliferation and differentiation of preosteoblasts (p < 0.001) and several pathways related to carbohydrate metabolism and cell signaling were suppressed. However, silencing of GLUT3 resulted in increased proliferation (p < 0.001), despite suppression of several pathways involved in cellular metabolism, biosynthesis and actin organization. Silencing of GLUT1 had no effect on proliferation and less changes in the transcriptome. RNA-seq dataset further revealed that osteoblasts express also class II and III glucose transporters, except for GLUT7. In conclusion, GLUT1, -3 and -4 may all contribute to glucose uptake in differentiating osteoblasts. GLUT4 expression was clearly required for osteoblast proliferation and differentiation. GLUT1 appears to be abundant in early precursors, but stable expression of GLUT3 suggest also a role for GLUT3 in osteoblasts. Presence of other GLUT members may further contribute to fine-tuning of glucose uptake. Together, glucose uptake in osteoblast lineage appears to rely on several glucose transporters to ensure sufficient energy for new bone formation.

Biological response to an experimental implant for tibial tuberosity advancement in dogs: A pre-clinical study.

In this study, we propose a novel bioresorbable bioactive implant for tibial tuberosity advancement (TTA). The implant consists of a gradually resorbing load-bearing shell which encompasses rapidly resorbing small casings loaded with silica-based bioactive glass (BG) particulates which promote bone formation and reduce the risk of infection. The shell and the casings are manufactured by 3D printing from two medical grade bioresorbable polymers (a polyglycolide/lactide based and a polydioxanone based) that have different degradation rates. The casings are expected to resorb within days after surgery to expose the BG particulates while the shell would retain the load-bearing properties of the implant for the time required by bone healing. Unlike the currently used metallic devices, the novel implant is resorbed and excreted from the body once its purpose is fulfilled. This study presents a logical progression from the in vitro characterisation of the materials and implants to the in vivo investigation of the experimental implants. This included mechanical testing of the materials, finite element analysis of a preliminary design of the novel TTA implant, assessment of the degradation behaviour of the polymers and the ion exchange of BG in simulated body fluid, and investigation of the biological response to the novel implants after implantation in rabbits. The osteointegration of the novel implants was comparable to the osteointegration of Ti6Al4V implants in the control group; the biological efficacy and safety were confirmed. The biological response was in line with the expectations. The proof of concept for the novel TTA implants was demonstrated.