Oxygen-releasing biomaterials for osteoarthritis: advances in managing the hypoxic joint microenvironment

Solid tumors often contain hypoxic microenvironments due to abnormal vasculatures and outweighing demands of oxygen. Cancer cells rely on anaerobic respiration, leading to sequential acidic microenvironments. Hypoxic and acidic microenvironments cause genetic instability and activate signaling pathways, contributing to cancer progression and therapy resistance, and have become targets for developing novel anti-cancer agents. This article reviews recent advances in the development of novel anti-cancer drugs targeting hypoxic and acidic microenvironments. Recent patents and published literature related to anti-cancer agents targeting tumor hypoxic and acidic microenvironments were searched and reviewed. Key termed used in the searching included cancer, anti-cancer drug, neoplasm, clinical trials, tumor microenvironment, hypoxic microenvironment, acidic microenvironment, hypoxia-inducible factors, hypoxia; metabolism; Warburg effect and aerobic glycolysis. A number of Hypoxia-Inducible Factor (HIF) inhibitors have been developed or discovered, but most of them have only exhibited indirect effects on HIFs, and a limited number of drugs are able to directly interfere with mRNA and protein of HIFs, the dimerization of α and β subunits, or the interaction between HIFs and its activators. The development of agents targeting acidic microenvironments focuses on V-ATPase, monocarboxylic acid transporters, Na+/H+ exchangers and carbonic anhydrases. Proton pump inhibitors as V-ATPase inhibitors have been applied in treating various tumors as an adjuvant therapy, but none of the other inhibitors has been approved for cancer treatment. Developing more specific agents, and seeking sensitive, applicable and accurate biomarkers may improve the efficacy of drugs targeting hypoxic and acidic microenvironments.

The transient receptor potential vanilloid 4 ion channel regulates the biosynthetic response of chondrocytes to dynamic loading

Osteoarthritis (OA) is a degenerative joint disease and is characterized by articular cartilage degeneration, subchondral bone sclerosis, and osteophyte formation with major clinical symptoms, including chronic pain, joint instability, stiffness, and radiographic joint space narrowing.1 OA is the most common form of arthritis and is a leading cause of impaired mobility in the elderly. It has been forecast that 25% of the adult population, or >50 million people in the United States, will be affected by OA by the year 2020, and it will be a major cause of morbidity and physical limitation among individuals older than 40 years. In addition to the well-documented impact of OA on physical function and quality of life, depression, and anxiety, there is a significant financial burden, with aggregate annual medical expenditures exceeding $185 billion in 2008.2 A variety of risk factors has been identified in the initiation and/or progression of OA, including age, gender, traumatic injury, obesity, metabolic dysfunction, and environmental and genetic factors.1 Despite extensive research over the past 20 years to delineate the pathogenic mechanism or mechanisms of OA, a full understanding of the initiators of the disease and the factors that accelerate OA progression is yet to be achieved. Thus, there is no clinical diagnosis for early OA and no effective disease-modifying treatment of late OA other than pain-relieving medication or the replacement of damaged joints.1 Normal articular cartilage that emerges during the postnatal stage as a permanent tissue distinct from the growth plate cartilage is a smooth, hard, white tissue that lines the surface of all diarthrodial joints. Collagens and proteoglycans are the principle extracellular matrix (ECM) molecules of articular cartilage. Mutations of ECM-related factors, including types II, IX, and XI collagen, have been reported in OA patients.1 Articular chondrocytes are the cells responsible for the maintenance of articular cartilage. As such, the dysregulation of this cell is directly connected to the process of cartilage degeneration in OA. Thus, understanding the phenotypic behavior of articular chondrocytes in homeostasis and disease has made us aware of several key environmental and genetic factors that affect OA initiation and progression. In earlier decades, the surgically induced destabilization of medial meniscus model, as well as genetic mouse models, were developed and demonstrated potential roles of affected genes in OA pathogenesis. Transforming growth factor (TGF)-β/Smad, Wnt/β-catenin, Notch, and Indian hedgehog pathways have demonstrated the critical and unique roles of chondrocytes during OA development and progression by stimulating chondrocytes toward hypertrophy.1 Recent genetic findings further suggest that Runx2, Mmp13, and Adamts5 are common target genes involved in the above-mentioned signaling networks to disrupt the metabolic and catabolic balance in chondrocytes; they eventually degrade cartilage matrix by upregulation of matrix metalloproteinases and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) activity and by downregulation of type II collagen and aggrecan synthesis.1 Recent studies of genome-wide association screens have been performed in large numbers of OA and control populations throughout the world and have confirmed several critical signaling molecules previously implicated by mouse genetic and injury-induced animal models, including the Wnt (Sfrp3), bone morphogenetic protein (Gdf5), and TGF-β (Smad3) signaling pathways.1 In addition to the single-nucleotide polymorphisms analysis, growing evidence points to the fact that the gene expression profile can be largely regulated by epigenetic machinery, modulating the local transcriptional activity and manipulating mRNA expression through a microRNA-mediated regulatory mechanism.3 Recent genome-wide methylation screening in patients with OA revealed different DNA methylation signatures in both synoviocytes and chondrocytes, indicating that epigenetic changes can influence OA susceptibility and severity.4 Epigenetic modification of Mmp13 and Adamts5 was also observed during OA development and progression, indicating that epigenetic factors may also play a role in the pathophysiology of OA.3 In addition to articular chondrocytes, other cell types, such as the mesenchymal stem cell in subchondral bone and synovial fibroblasts, contribute to OA progression. TGF-βs, in response to abnormal mechanical loading, were found to be released, activated, and accumulated in the subchondral bone in patients with OA, leading to aberrant bone formation and angiogenesis through recruitment of osteoprogenitor cells.5 Both injury and obesity-induced low-grade inflammation have been widely recognized as contributing factors to synovial tissue expansion and to hyperplasia in the early onset of OA. The inflammatory factors and ECM-degrading enzymes facilitate OA progression.6 As these underlying mechanisms are further delineated, manipulation of several critical molecules could serve as potential key targets for therapeutic intervention for the treatment of OA disease.

Epithelial-mesenchymal transition (EMT) is a normal developmental process by which epithelial cells lose their polarity and cell-cell adhesions to become more motile and invasive mesenchymal cells. EMT is associated with the earliest stages in metastasis and promotes chemoresistance in a variety of carcinomas, including pancreatic ductal adenocarcinoma (PDAC). While specific growth factors (e.g., transforming growth factor beta) are perhaps the most well studied inducers of EMT, the low oxygen tension (hypoxia) characteristic of the PDAC tumor microenvironment has also been reported to drive EMT. The signaling pathways that promote EMT in the hypoxic PDAC tumor microenvironment may therefore represent useful drug targets for combination therapy approaches as adjuvants to augment the efficacy of chemotherapy. However, hypoxia-driven EMT has not been well characterized in PDAC, nor have the relevant hypoxia-driven signaling pathways been identified. Our lab recently developed a statistical model of the multivariate signaling regulation of growth factor-induced EMT to identify that the ERK, JNK, and NF-kappaB pathways coordinate to drive EMT in PDAC cells. In the present study, we sought to determine the roles of those pathways in promoting EMT in PDAC cells in response to hypoxia. We created an in vitro model of hypoxia-driven EMT by culturing PDAC cells in 1% oxygen and characterizing dynamic changes in the expression of hypoxia inducible factors (HIF) 1 and 2 alpha, as well as the concomitant loss of E-cadherin protein and transcripts, increased abundance of vimentin-positive cells, and increased expression of the mesenchymal transcripts VIM, SNAI1, and SNAI2. Hypoxia-driven EMT was observed in human PDAC cell lines, patient-derived xenograft cell lines, and KPC murine cell lines. To probe for the activation of specific signaling pathways, we used a combination of immunoblotting and immunofluorescence microscopy. Automated image analysis of immunofluorescence images was used to understand the relevance of specific signaling pathways for explaining the heterogeneity of EMT within populations of cells. We further identified potential kinase targets for antagonizing EMT in the hypoxic microenvironment and confirmed their roles by pharmacological inhibition and/or siRNA-mediated knockdown. We also found that specific kinase pathways are involved in the stabilization of HIF proteins in hypoxia. The relevance of these pathways in driving EMT is being further validated through analyses of a patient-derived xenograft mouse model of PDAC and a HIF1A deletion/Kras mutant autochthonous mouse model of PDAC. Citation Format: Brooke A. McGirr, Nicholas M. Seyler, Matthew J. Lazzara. Signaling regulation of epithelial-mesenchymal transition in the hypoxic tumor microenvironment of pancreas cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-055.

ABSTRACT
 MESENCHYMAL STEM CELL THERAPY AS OSTEOARTHRITIS TREATMENT MODALITY
 Introduction: Osteoarthritis (OA) is a degenerative and progressive disease with articular cartilage degradation, osteophyte formation, subcondral bone damage, and inflammation of the synovial membrane. The incidence of joint disease aged over 15 years in Indonesia as much as 15%, with 60% is OA in the knee. Recent research mentions the potential for mesechymal stem cell (MSC) as OA treatment.
 Discussion: The pathogenesis of OA involves activation of the immune system which causes a decrease in cartilage synthesis and cartilage damage. The immune system also triggers the activation of c-Jun NH 2-terminal Kinase (JNK) signaling pathway which causes cartilage degeneration. OA treatment has evolved into intra-articular therapy, one of which is mesenchymal stem cell (MSC). The mechanism of MSC in OA therapy is to stimulate type two collagen for cartilage regeneration, accelerate cartilage differentiation, and have anti-inflammatory effects. The use of MSC by autotransplantation can be taken through adipose tissue liposuction and bone marrow aspiration. The best use of allotransplantation MSC comes from Wharthon’s Jelly cells from human umbilical cord tissue (HUCT).
 Conclusion: Potency of mesechymal stem in cell cartilage’s tissue regeneration process and anti-inflammatory after oxidative stress exposure could be developed as a novel therapy modality to prevent worsening of the disease of patients with OA.
 Keywords: Osteoarthritis, Stem cell, Therapy

Background Knee osteoarthritis (KOA) is characterized by cartilage degradation, osteophyte formation, and synovitis. Cartilage degradation in KOA begins with the loss of aggrecan, primarily due to A Disintegrin and Metalloproteinase with Thrombospondin Motif 5 (ADAMTS5), which is produced by chondrocytes and synovial cells and a key target for therapeutic intervention. Current treatments for KOA primarily focus on pain relief, as disease-modifying osteoarthritis drugs (DMOADs) remain unavailable. Electroacupuncture (EA), applying electrical stimulation to acupoints, has been investigated for its potential to alleviate KOA symptoms; however, the specific effects of different acupoint combinations remain unclear. This study investigates the effect of EA on pain and cartilage degeneration in a KOA rat model by examining ADAMTS5 expression in synovial tissue. Materials and methods Male Wistar rats were divided into five groups: control, sham-operated, KOA model, KOA treated with EA at ST36 (Zusanli)-LR8 (Ququan) (KOA+LR8), and KOA treated at ST36-Ex-LE2 (Heding) (KOA+Ex-LE2). The DMM (destabilization of the medial meniscus) procedure induced KOA, and EA was applied thrice weekly for four weeks. The rotarod test was used to assess motor coordination, and samples were collected for immunofluorescence, Western blot, and histological analysis. Pain was assessed via c-fos expression in the spinal cord, while Safranin O-Fast Green staining was used to evaluate cartilage degeneration via the Osteoarthritis Research Society International (OARSI) scoring system. Results The KOA group post-surgery showed reduced motor coordination, while EA at both ST36-LR8 and ST36-Ex-LE2 enhanced performance (day 28: control: 28.8 ± 0.6, sham: 28.4 ± 3.7, KOA: 19.7 ± 0.9, KOA+LR8: 24.8 ± 1.5, KOA+Ex-LE2: 26.9 ± 1.2). Expression of c-fos, elevated in the KOA group, was significantly suppressed by EA (control: 7.6 ± 0.9, sham: 13.6 ± 2.8, KOA: 24.5 ± 2.1, KOA+LR8: 12.8 ± 0.9, KOA+Ex-LE2: 17.0 ± 1.2). Histologically, KOA rats showed severe cartilage degradation and osteophyte formation, while EA at ST36-Ex-LE2 significantly reduced these changes (control: 0.2 ± 0.1, sham: 0.4 ± 0.2, KOA: 1.8 ± 0.4, KOA+LR8: 1.0 ± 0.2, KOA+Ex-LE2: 0.5 ± 0.2). The ST36-LR8 group also showed improvements, although less pronounced than the ST36-Ex-LE2 group. Western blotting revealed that DMM-induced ADAMTS5 expression was significantly inhibited by EA at ST36-Ex-LE2 but not at ST36-LR8 (control: 1.0 ± 0, sham: 1.2 ± 0.4, KOA: 3.0 ± 0.3, KOA+LR8: 2.1 ± 0.3, KOA+Ex-LE2: 1.4 ± 0.4). Conclusion EA at ST36-Ex-LE2 showed a remarkable protective effect on articular cartilage by inhibiting ADAMTS5 expression from synovium, suggesting that it can break the vicious cycle of synovitis and cartilage destruction. In contrast, EA at ST36-LR8 had a moderate effect on cartilage degeneration and ADAMTS5 expression. The difference in efficacy may be due to the anatomical differences between acupoints. ST36-Ex-LE2 coincides with an area rich in synovial fibroblasts and mast cells involved in inflammation and pain. This highlights the importance of acupoint selection to maximize the therapeutic effect of EA. The specificity of this acupoint combination provides a potential strategy for managing KOA and slowing the progression of the disease. Further studies are needed to elucidate the detailed mechanisms behind the effects of EA and explore its potential as an alternative or complementary treatment for KOA.

PPARdelta promotes the progression of post-traumatic osteoarthritis

Disclosure: J. Rakholiya: None. S. Nagaraj: None. P. Sharma: None. Introduction: Alkaline phosphatase (ALP) elevation is commonly seen in hepatobiliary disorders. Conditions of high turnover such as healing fractures, hyperparathyroidism, Pagets’ disease of the bone, also lead to ALP elevation (1). Osteoarthritis (OA), characterised by degeneration of articular cartilage, joint space narrowing, and osteophyte formation, rarely causes ALP elevation. We present a case of ALP elevation that resolved after the arthritic joint was replaced. Case Presentation: A 60-year-old female with history of bilateral knee OA presented to the endocrinology clinic due to elevated ALP for over a year. She reported chronic fatigue, sleep disturbance, dry skin, hair loss, inability to lose weight and anxiety. ALP level at presentation was 165 U/L. An extensive work-up showed unremarkable serum and ionized calcium, serum phosphorus, parathyroid hormone, vitamin D 25-OH, calcitriol, 24-hour urine calcium, serum protein electrophoresis, and 24-hour urine protein electrophoresis. Dual x-ray absorptiometry (DXA) scan was normal with Fracture risk assessment tool (FRAX) score 5.9% for major osteoporotic fracture and 0.2% for hip fracture. Patient underwent total left knee arthroplasty following which ALP improved from 165 U/L to 135 U/L within 9 weeks of the procedure. Five months later, ALP was elevated (164 U/L) again. She then underwent right knee arthroplasty and ALP dropped to normal level (114 U/L) within 1 week post-surgery. Discussion: We report the rare phenomenon of ALP elevation in severe OA, that resolved after joint replacement. OA is characterised by inflammation mediated cartilage destruction, subchondral bone change, osteophyte formation, and alterations of ligaments and meniscuses. Bone change in severe OA is associated with increased synovial fluid ALP level. However, there is a paucity of literature studying the association of ALP with OA. A cross-sectional study of 3060 patients demonstrated higher serum ALP with symptomatic knee OA. Another study showed higher ALP levels with increased joint stiffness. The ALP elevation was attributed to low grade inflammation causing an inflammatory response in chondrocytes, and osteoarthritic pain. There is a need for more studies to evaluate the utility of serum ALP as a biomarker for severe OA. Presentation: Thursday, June 15, 2023

Identification and analysis of key microRNAs derived from osteoarthritis synovial fluid exosomes.

Hypoxia-inducible factors: where, when and why?

Harnessing knee joint resident mesenchymal stem cells in cartilage tissue engineering.

Background: The clinical applications of stromal vascular fraction (SVF) therapy for osteoarthritis (OA) have attracted academic and clinical attention. However, data of the effects of stromal vascular fraction therapy on regeneration of degenerated cartilage are limited in the literature. Meanwhile, there is a great need for a simple and non-invasive evaluation method to analyze the changes of joint cartilage qualitatively and quantitatively in clinical trials. This study entitled “stromal vascular fraction Therapy for Human Knee Osteoarthritis” was registered in ClinicalTrial.gov # NCT05019378.Materials and Methods: We designed and conducted a single center, open labeled clinical phase I/II study, and 6 osteoarthritis patients with both knee cartilage defect I-II were enrolled in this study. The two knees of each patient were randomly assigned to autologous stromal vascular fraction treatment group or non-treatment control group to evaluate the safety and therapeutic effect of stromal vascular fraction therapy for human knee osteoarthritis. We have also established a novel protocol to provide 3D MRI imaging for human knee cartilage enabling us to qualitatively and quantitatively evaluate cartilage degeneration and regeneration in this study.Results: The qualitative and quantitative evaluation of 3D Magnetic Resonance Imaging (MRI) imaging of knee cartilage demonstrated that the stromal vascular fraction therapy reduced the cartilage defects; and significant increase of cartilage value both in defect cartilage area and whole cartilage area of treated group and significant increase of thickness and area of both femoral and tibia cartilage in vertical sections of the stromal vascular fraction treated Group at 12 and 24 W post treatment in cartilage defect I-II osteoarthritis patients.Conclusion: This clinical phase I/II study indicated that stromal vascular fraction therapy is a safe clinical procedure and provided evidence that the stromal vascular fraction therapy significantly facilitated cartilage regeneration, opening the opportunity to a phase III trial investigating authentic efficacy of the procedure. This study is the first qualitative and quantitative evaluation of the efficacy of autologous stromal vascular fraction cellular therapy on cartilage regeneration. Through early and definite diagnosis of knee osteoarthritis patients, and providing safe and efficient therapy to facilitate cartilage regeneration, we will be able to control or reverse cartilage degeneration and completely change the epidemiology of osteoarthritis worldwide.

Osteoarthritis (OA) is a chronic degenerative disease characterized by joint degradation and severe pain, with no effective clinical treatments currently available. Biomaterials, due to their functional and structural diversity, hold significant clinical translation potential to meet various needs in OA management. However, the development of biomaterial scaffold strategies must align closely with advancements in the understanding of OA pathophysiology. This review revisits the interplay between aging, hypoxic microenvironments, and OA progression from a novel perspective. It details the aging-related phenotypes characterized by impaired autophagy and inflammation in various OA-affected tissues and introduces NDRG-mediated hypoxia regulation mechanisms that function independently of the hypoxia-inducible factor (HIF) family. Additionally, it systematically summarizes biomaterial scaffold strategies for OA treatment, including hydrogels, nanoparticles, microneedles, and exosomes. These strategies, inspired by new insights, aim to enhance drug bioavailability, reduce systemic effects, prolong therapeutic duration, and ameliorate the adverse microenvironment of OA through multiple modes of action. This approach holds promise for providing novel ideas for microenvironment-targeted interventions in other degenerative diseases.

Phosphocitrate reduced cartilage degeneration in non-calcification induced osteoarthritis

Bone–cartilage interface crosstalk in osteoarthritis: potential pathways and future therapeutic strategies