Glycolytic reprogramming in macrophages and MSCs during inflammation.

Background: Dysregulated inflammation is associated with many skeletal diseases and disorders, such as osteolysis, non-union of fractures, osteonecrosis, osteoarthritis and orthopaedic infections. We previously showed that continuous infusion of lipopolysaccharide (LPS) contaminated polyethylene particles (cPE) caused prolonged inflammation and impaired bone formation. However, the metabolic and bioenergetic processes associated with inflammation of bone are unknown. Mitochondria are highly dynamic organelles that modulate cell metabolism and orchestrate the inflammatory responses that involve both resident and recruited cells. Glycolytic reprogramming, the shift from oxidative phosphorylation (OXPHOS) to glycolysis causes inappropriate cell activation and function, resulting in dysfunctional cellular metabolism. We hypothesized that impaired immunoregulation and bone regeneration from inflammatory states are associated with glycolytic reprogramming and mitochondrial dysfunction in macrophages (Mφ) and mesenchymal stromal cells (MSCs). Methods: We used the Seahorse XF96 analyzer and real-time qPCR to study the bioenergetics of Mφ and MSCs exposed to cPE. To understand the oxygen consumption rate (OCR), we used Seahorse XF Cell Mito Stress Test Kit with Seahorse XF96 analyzer. Similarly, Seahorse XF Glycolytic Rate Assay Kit was used to detect the extracellular acidification rate (ECAR) and Seahorse XF Real-Time ATP Rate Assay kit was used to detect the real-time ATP production rates from OXPHOS and glycolysis. Real-time qPCR was performed to analyze the gene expression of key enzymes in glycolysis and mitochondrial biogenesis. We further detected the gene expression of proinflammatory cytokines in Mφ and genes related to cell differentiation in MSC during the challenge of cPE. Results: Our results demonstrated that the oxidative phosphorylation of Mφ exposed to cPE was significantly decreased when compared with the control group. We found reduced basal, maximal and ATP-production coupled respiration rates, and decreased proton leak in Mφ during challenge with cPE. Meanwhile, Mφ showed increased basal glycolysis and proton efflux rates (PER) when exposed to cPE. The percentage (%) of PER from glycolysis was higher in Mφ exposed to cPE, indicating that the contribution of the glycolytic pathway to total extracellular acidification was elevated during the challenge of cPE. In line with the results of OCR and ECAR, we found Mφ during cPE challenge showed higher glycolytic ATP (glycoATP) production rates and lower mitochondrial ATP (mitoATP) production rates which is mainly from OXPHOS. Interestingly, MSCs showed enhanced glycolysis during challenge with cPE, but no significant changes in oxygen consumption rates (OCR). In accordance, seahorse assay of real-time ATP revealed glycoATP rates were elevated while mitoATP rates showed no significant differences in MSC during challenge with cPE. Furthermore, Mφ and MSCs exposed to cPE showed upregulated gene expression levels of glycolytic regulators and Mφ exposed to cPE expressed higher levels of pro-inflammatory cytokines. Conclusion: This study demonstrated the dysfunctional bioenergetic activity of bone marrow-derived Mφ and MSCs exposed to cPE, which could impair the immunoregulatory properties of cells in the bone niche. The underlying molecular defect related to disordered mitochondrial function could represent a potential therapeutic target during the resolution of inflammation.

Managing early complications in total hip arthroplasty: the safety of immediate revision.

PURPOSE: Immediate revision refers to a reoperation that involves resetting, draping, and exchanging the implant, after wound closure in total hip arthroplasty. The purpose of this study is to investigate the impact of immediate revision after total hip arthroplasty on subsequent infection and complication rates. METHODS: A total of 14,076 primary total hip arthroplasties performed between 2010 and 2020 were identified in our institutional database, of which 42 underwent immediate revision. Infection rates were determined 2 years after the index arthroplasty. The cause and type of revision, duration of primary and revision surgeries, National Nosocomial Infections Surveillance score, implant type, changes in implants, complications, and preoperative and intraoperative antibiotic prophylaxis were all determined. RESULTS: No infections were observed within 2 years after the index arthroplasty. Leg length discrepancy (88%, n = 37) and dislocation (7.1%, n = 3) were the main causes of immediate revision. In most cases of discrepancy, the limb was clinically and radiologically longer before the immediate revision. The mean operative time was 48 ± 14 min for the primary procedure and 23.6 ± 9 min for the revision. The time between the first incision and last skin closure ranged from 1 to 3 h. None of the patients were extubated between the two procedures. Two patients had a National Nosocomial Infections Surveillance score of 2, 13 had a score of 1, and 27 had a score of 0. CONCLUSION: Immediate revision is safe for correcting clinical and radiological abnormalities, and may not be associated with increased complication or infection rates. STUDY DESIGN: Retrospective cohort study; level of evidence, 3.

Metabolic profile of mesenchymal stromal cells and macrophages in the presence of polyethylene particles in a 3D model.

BACKGROUND: Continuous cross talk between MSCs and macrophages is integral to acute and chronic inflammation resulting from contaminated polyethylene particles (cPE); however, the effect of this inflammatory microenvironment on mitochondrial metabolism has not been fully elucidated. We hypothesized that (a) exposure to cPE leads to impaired mitochondrial metabolism and glycolytic reprogramming and (b) macrophages play a key role in this pathway. METHODS: We cultured MSCs with/without uncommitted M0 macrophages, with/without cPE in 3-dimensional gelatin methacrylate (3D GelMA) constructs/scaffolds. We evaluated mitochondrial function (membrane potential and reactive oxygen species-ROS production), metabolic pathways for adenosine triphosphate (ATP) production (glycolysis or oxidative phosphorylation) and response to stress mechanisms. We also studied macrophage polarization toward the pro-inflammatory M1 or the anti-inflammatory M2 phenotype and the osteogenic differentiation of MSCs. RESULTS: Exposure to cPE impaired mitochondrial metabolism of MSCs; addition of M0 macrophages restored healthy mitochondrial function. Macrophages exposed to cPE-induced glycolytic reprogramming, but also initiated a response to this stress to restore mitochondrial biogenesis and homeostatic oxidative phosphorylation. Uncommitted M0 macrophages in coculture with MSC polarized to both M1 and M2 phenotypes. Osteogenesis was comparable among groups after 21 days. CONCLUSION: This work confirmed that cPE exposure triggers impaired mitochondrial metabolism and glycolytic reprogramming in a 3D coculture model of MSCs and macrophages and demonstrated that macrophages cocultured with MSCs undergo metabolic changes to maintain energy production and restore homeostatic metabolism.

The efficiency of genetically modified mesenchymal stromal cells combined with a functionally graded scaffold for bone regeneration in corticosteroid-induced osteonecrosis of the femoral head in rabbits.

Core decompression (CD) with mesenchymal stromal cells (MSCs) is an effective therapy for early-stage osteonecrosis of the femoral head (ONFH). Preconditioning of MSCs, using inflammatory mediators, is widely used in immunology and various cell therapies. We developed a three-dimensional printed functionally graded scaffold (FGS), made of ß-TCP and PCL, for cell delivery at a specific location. The present study examined the efficacy of CD treatments with genetically modified (GM) MSCs over-expressing PDGF-BB (PDGF-MSCs) or GM MSCs co-over-expressing IL-4 and PDGF-BB and preconditioned for three days of exposure to lipopolysaccharide and tumor necrosis factor-alpha (IL-4-PDGF-pMSCs) using the FGS for treating steroid-induced ONFH in rabbits. We compared CD without cell-therapy, with IL-4-PDGF-pMSCs alone, and with FGS loaded with PDGF-MSCs or IL-4-PDGF-pMSCs. For the area inside the CD, the bone volume in the CD alone was higher than in both FGS groups. The IL-4-PDGF-pMSCs alone and FGS + PDGF-MSCs reduced the occurrence of empty lacunae and improved osteoclastogenesis. There was no significant difference in angiogenesis among the four groups. The combined effect of GM MSCs or pMSCs and the FGS was not superior to the effect of each alone. To establish an important adjunctive therapy for CD for early ONFH in the future, it is necessary and essential to develop an FGS that delivers biologics appropriately and provides structural and mechanical support.

Unplanned return to the operating room after arthroscopic procedures: a need to consider 12 months after the initial surgery.

INTRODUCTION: The purpose of this study was to report the proportion and cause of unplanned revision surgery rates within 1 year following arthroscopic procedures. Our hypothesis was that there is a significant rate of unplanned returns (URs) occurring between 3 and 12 months after the initial procedure and that causes of revision are different when considering the delay after the index surgery. MATERIALS AND METHODS: Among 4142 consecutive patients who underwent an arthroscopic procedure in a single department of orthopedics and traumatology, patients undergoing revision surgery for any reasons directly related to the primary procedure were included. Cause for revision, surgical site, delay from index procedure, and number of revisions were screened. RESULTS: Seventy-eight patients underwent 97 revision surgeries (2.3%) for reasons directly related to the primary procedure. Most revision surgeries were performed after month 3 following index surgery (59 patients, 60.8%). Mean time to revision surgery was 5.3 ± 4.3 months (range 0-365 days). Usual early-onset (< 3 months) reasons for unplanned revision were surgical site infection (17 patients, 0.41%), wound-healing defect (12 patients, 0.29%), and hemorrhagic complication (7 patients, 0.17%). Reasons for delayed unplanned revision (> 3 months) were index procedure failure (21 patients, 0.51%), stiffness (18 patients, 0.43%), and removal of hardware (16 patients, 0.41%). CONCLUSIONS: Reasons for return to the operating room (OR) are different depending on the timepoint from index procedure. Patients should receive relevant information accordingly when scheduling any arthroscopic procedure, including up to 1-year potential complications. LEVEL OF EVIDENCE: Prognostic study, Case series, Level IV.

Therapeutic effects of MSCs, genetically modified MSCs, and NFĸB-inhibitor on chronic inflammatory osteolysis in aged mice.

The number of total joint replacements is increasing, especially in elderly patients, and so too are implant-related complications such as prosthesis loosening. Wear particles from the prosthesis induce a chronic inflammatory reaction and subsequent osteolysis, leading to the need for revision surgery. This study investigated the therapeutic effect of NF-ĸB decoy oligodeoxynucleotides (ODN), mesenchymal stem cells (MSCs), and genetically-modified NF-ĸB sensing interleukin-4 over-secreting MSCs (IL4-MSCs) on chronic inflammation in aged mice. The model was generated by continuous infusion of contaminated polyethylene particles into the intramedullary space of the distal femur of aged mice (15-17 months old) for 6 weeks. Local delivery of ODN showed increased bone mineral density (BMD), decreased osteoclast-like cells, increased alkaline phosphatase (ALP)-positive area, and increased M2/M1 macrophage ratio. Local injection of MSCs and IL4-MSCs significantly decreased osteoclast-like cells and increased the M2/M1 ratio, with a greater trend for IL4-MSCs than MSCs. MSCs significantly increased ALP-positive area and BMD values compared with the control. The IL4-MSCs demonstrated higher values for both ALP-positive area and BMD. These findings demonstrated the therapeutic effects of ODN, MSCs, and IL4-MSCs on chronic inflammatory osteolysis in aged mice. The two MSC-based therapies were more effective than ODN in increasing the M2/M1 macrophage ratio, reducing bone resorption, and increasing bone formation. Specifically, MSCs were more effective in increasing bone formation, and IL4-MSCs were more effective in mitigating inflammation. This study suggests potential therapeutic strategies for treating wear particle-associated inflammatory osteolysis after arthroplasty in the elderly.

Sex differences in the therapeutic effect of unaltered versus NFκB sensing IL-4 over-expressing mesenchymal stromal cells in a murine model of chronic inflammatory bone loss.

Wear particles from joint arthroplasties induce chronic inflammation associated with prolonged upregulation of nuclear factor kappa-B (NF-κB) signaling in macrophages and osteoclasts, which leads to osteolysis and implant loosening. Mesenchymal stromal cell (MSC)-based therapy showed great potential for immunomodulation and mitigation of osteolysis in vivo, especially in the chronic phase of inflammation. We previously generated genetically modified MSCs that secrete the anti-inflammatory cytokine interleukin 4 (IL-4) in response to NF-κB activation (NFκB-IL-4 MSCs). However, whether the impact of sexual difference in the internal environment can alter the therapeutic effects of IL-4 over-secreting MSCs that simultaneously mitigate prolonged inflammation and enhance bone formation remains unknown. This study investigated the therapeutic effects of unaltered MSCs versus NFκB-IL-4 MSCs in mitigating chronic inflammation and enhancing bone formation in male and female mice. The murine model was established by continuous infusion of polyethylene particles contaminated with lipopolysaccharide (cPE) into the medullary cavity of the distal femur for 6 weeks to induce chronic inflammation. Unaltered MSCs or NFκB-IL-4 MSCs were infused into the femoral intramedullary cavity in sex-matched groups beginning 3 weeks after primary surgery. Femurs were harvested at 6 weeks, and bone marrow density was measured with micro-computational tomography. Numbers of osteoclast-like cells, osteoblasts, and macrophages were evaluated with histochemical and immunofluorescence staining. cPE infusion resulted in severe bone loss at the surgery site, increased tartrate-resistant acid phosphatase positive osteoclasts and M1 pro-inflammatory macrophages, and decreased alkaline phosphatase expression. MSC-based therapy effectively decreased local bone loss and polarized M1 macrophages into an M2 anti-inflammatory phenotype. In females, unaltered MSCs demonstrated a larger impact in enhancing the osteogenesis, but they demonstrated similar anti-inflammatory effects compared to NFκB-IL-4 MSCs. These results demonstrated that local inflammatory bone loss can be effectively modulated via MSC-based treatments in a sexually dimorphic manner, which could be an efficacious therapeutic strategy for treatment of periprosthetic osteolysis in both genders.

Importance of Early Diagnosis and Care in Knee Dislocations Associated with Vascular Injuries.

BACKGROUND: Arterial injury secondary to acute knee dislocation (KD) is a rare but devastative complication. The aim of this study is to evaluate functional sequelae and factors of poor prognosis. METHODS: A retrospective monocentric series of consecutive KD with acute ischemia by popliteal artery injury was analyzed between 2005 and 2017. The main outcome was the amputation rate. RESULTS: Sixteen dislocations were included. Nine (56%) were due to public road accidents, 5 (31%) were due to falls from height, and 2 (13%) were due to sports injuries. Dislocation had occurred in the posterior location in 8 (50%) cases. Regarding arterial injury, there were 7 (44%) ruptures, 7 (44%) dissections, and 2 (13%) isolated thromboses. Eleven (69%) KDs with vascular trauma were associated with signs of acute ischemia. Revascularization was achieved by anatomical venous bypass in 14 (88%), resection and direct anastomosis in one (6%), and isolated thrombectomy in one (6%). Median time to surgery (time between trauma and vascular repair) was 7 hours (3.25-60.92 hours). Primary revascularization was performed in 12 (75%) cases. In three cases (19%), orthopedic reduction and stabilization were performed first. In one case, (6%) three-step management with vascular shunt at first, then with knee stabilization, and finally vascular bypass was carried out. Stabilization was achieved by using an external fixator in 13 (82%) cases, by open reduction and internal fixation in one case (6%), by ligamentoplasty in one (6%), and by using a long leg cast in one (6%). Fasciotomy was required in 12 (75%) cases. Two patients had early vascular complications, and 2 had early systemic complications. Three secondary transfemoral amputations were performed. Median follow-up duration was 23 months. No secondary amputation was recorded. At the end of follow-up, functional outcomes were evaluated using the Oxford Knee Score (OKS). The median OKS was 30 versus the pretrauma median OKS of 47 (P < 0.00028). No risk factor associated with limb amputation has been highlighted. CONCLUSIONS: Analysis of these results provided indications for therapeutic management of this condition. This study shows poor functional outcomes because of severity of vascular lesion in patients with orthopedic trauma but with healthy arteries.