Dongrim Seol

Intra-articular injection of autologous Platelet Rich-Platelet Plasma (PRP) is commonly employed as a non-surgical option for treating early-stage knee osteoarthritis (OA). Placebo-controlled clinical trials show sustained improvements in knee pain and function in most, but not all patients. Although several hypotheses have been put forward, the reasons for inconsistent responses remain unclear. Exosomes, a class of lipid membrane-bound extracellular otherwise known as small extracellular vesicles (sEVs), are a prominent component of PRP. sEVs are thought to mediate many of PRP’s therapeutic effects by carrying complex molecular cargos that regulate gene expression when taken up by cells in osteoarthritic joints. We hypothesized that patient-to-patient variations in sEV protein content correlate with patient responses to PRP. To test this, we compared the proteomes of PRP-derived sEVs from 3 patients who reported significant improvement in symptoms from baseline to 12 weeks post-injection (responders) versus 3 who did not (non-responders). 48 proteins identified by mass spectrometry were significantly different between Responders and Non-Responders. Taken together, these findings indicate that a patient’s responsiveness to PRP treatment could be associated with EV protein cargo composition.

Background: Intra-articular injections of autologous platelet-rich plasma (PRP) have been shown to reduce joint pain in many patients with mild to moderate knee osteoarthritis (OA) (PRP responders). However, for unclear reasons, 20% to 30% of patients experience little to no improvement in symptoms (PRP nonresponders). Understanding the mechanisms underlying PRP's action is crucial to address this limitation. In vitro and animal studies suggest that the benefits of PRP in knee OA are partly attributable to exosomes secreted by platelets and other cells. Purpose/Hypothesis: This study aimed to (1) determine the effects of intra-articular PRP injections in patients with symptomatic knee OA on pain and physical function, and (2) evaluate whether PRP exosome concentration and size were associated with therapeutic efficacy. Responsiveness to PRP therapy in patients with knee OA may be related to exosome concentration and size. Study Design: Case-control study; Level of evidence, 3. Methods: A total of 60 patients seeking nonsurgical treatment of symptomatic mild to moderate knee OA—Kellgren-Lawrence (KL) grades 1 to 3—were prospectively enrolled in the study. Baseline descriptive and health data were collected, including patient-reported pain and functional outcomes at baseline, 6 weeks, and 12 weeks after injection. PRP responders were defined as patients with ≥20% reduction in the Numerical Rating Scale (NRS) of joint pain at 12 weeks after treatment. Exosome size and concentration and platelet concentration were measured in each sample. Logistic regression was used to explore relationships between responder status and patient factors—age, body mass index (BMI), sex, and KL grade. The mean exosome size and concentration were also compared between responders and nonresponders using an independent t -test. Results: Of the 58 patients included in the analyses, 43 (73%) reported a 20% reduction in NRS scores. The mean exosome size among a subset of patients who reported unacceptable pain symptoms before treatment (n = 43) was 80.8 ± 2.7 nm for responders and 78.0 ± 1.5 nm for nonresponders, a statistically significant difference ( P = .005). There was no significant difference between responders and nonresponders in terms of platelet or exosome concentration. Age, BMI, sex, and KL grade were not significantly associated with the odds of PRP treatment response (all P > .05). Conclusion: PRP-derived exosome size appears to be associated with improvements in pain at 12 weeks after injection.

Reparative tertiary dentinogenesis requires the recruitment and odontogenic differentiation of dental pulp stem cells (DPSCs). Extracellular vesicles (EVs) as bioactive molecules have gained attention in regenerative medicine for their ability to mediate tissue repair through intercellular communication, influencing cell recruitment, proliferation, and differentiation. This study aimed to evaluate the effects of EVs on DPSC homing and odontogenic differentiation for dentin regeneration. DPSC-derived EVs were cultured in either growth (EV-G) or odontogenic differentiation (EV-O) conditions and isolated using a modified precipitation method. EVs were characterized by nanoparticle tracking analysis, scanning electron microscopy, antibody array, and cellular uptake assay. Treatment with 5 × 10⁸ EVs/mL significantly enhanced DPSC chemotaxis and proliferation compared with a no-treatment control and a lower dosage of EV (5 × 10⁷ EVs/mL). Gene expression and biochemical analyses revealed that EV-O up-regulated odontogenic markers including collagen type 1A1 (COL1A1), runt-related transcription factor 2 (RUNX2), and alkaline phosphatase (ALP). EV-O enhanced dentin regeneration by approximately 55% over vehicle controls in a rabbit partial dentinotomy/pulpotomy model. We identified key microRNAs (miR-21-5p, miR-221-3p, and miR-708-3p) in EV-O involved in cell homing and odontogenesis. In conclusion, our EV-based cell homing and odontogenic differentiation strategy has significant therapeutic potential for dentin regeneration.

Arthrofibrosis is defined as the excessive accumulation of connective tissue in and around joints, which interferes with the range of motion required for activities of daily living. Although joint stiffness can be restored by surgical interventions such as adhesion lysis, arthroscopic debridement, and capsular release, arthrofibrosis tends to redevelop in the months following the surgery. Thus, there is a critical and urgent need to develop a non-invasive, pharmacological therapy to prevent or resolve arthrofibrosis. A subclass of small extracellular vesicles called exosomes convey bioactive regulators like micro ribonucleic acids (miRNAs/miRs), which can function as anti- and pro-fibrotic agents. Currently, there is no research on miRNA-based therapeutic potentials for treating arthrofibrosis. Previous research and clinical observations on fibrosis across organ systems suggest that there are commonalities in pathogenic mechanisms that can be targeted in arthrofibrosis therapy. In this study, we collated and critically analyzed the existing literature on exosomal miRNAs in organ fibrosis to discover potential candidates for diagnosing, preventing, and/or treating arthrofibrosis. Fifty-six articles were finally selected and categorized by anti- and pro-fibrotic candidates of miRNAs. Notably, let-7, miR-26, miR-29, miR-146, miR-148/-152, miR-214, miR-223, and miR-21 emerged as prominent candidates that should be investigated further for effectiveness in arthrofibrosis therapy.

The activation of chondrogenic progenitor cells (CPCs) in articular cartilage during a traumatic injury is vital for cartilage regeneration. Although our understanding of the mechanisms underlying CPC chondrogenic activation remains incomplete, there is evidence that exosomal microRNAs (miRNAs or miRs) are involved in tissue healing due to their regulating role of posttranscriptional gene expressions. In this study, we profiled enriched and differential expression of miRNAs in exosomes derived from bovine joint cells (CPCs, chondrocytes, and synoviocytes) via Next Generation Sequencing analysis and validated the potential therapeutic effects of candidate exosomal miRNAs for cartilage regeneration. For CPC-based cartilage regeneration, we tested the impact of administering miR-107, miR-140, and miR-148a on CPCs because we found that these miRNAs were highly and differentially expressed in chondrocytes-derived exosomes (CC-Exo). We found that: (1) miR-140 induced chondrogenic gene expression including SRY-box transcription factor 9, collagen type 2A1, and aggrecan, and (2) miR-107 suppressed catabolic gene expression including matrix metalloproteinase 3, a disintegrin and metalloproteinase with thrombospondin motifs 5, and nitric oxide synthase 2. Our findings indicate that transfection of CPCs with specific chondrogenic miRNAs present in CC-Exo have the potential to promote CPC-based cartilage regeneration and could be an important component of posttraumatic osteoarthritis prevention.

Elbow trauma can lead to joint contracture and reduced range of motion (ROM). Nonsurgical interventions can improve ROM, but in some cases capsule release surgery is required. Although surgery can improve ROM, it often does not restore full ROM. Thus, alternatives are needed. One approach is to target activated myofibroblasts, which are commonly associated with fibrotic tissue. Mechanical and biochemical cues drive a feedback loop that can result in normal or pathological healing. We hypothesize that this feedback loop exists in joint contracture and can be manipulated so that myofibroblast activity is reduced, normal healing is achieved, and ROM is improved. We previously demonstrated that blebbistatin can inhibit myofibroblast contractile forces and reduce collagen synthesis in vitro. Thus, the purpose of this study was to assess the use of blebbistatin in an animal model of elbow contracture, which was induced in 7 groups of 4 rats each (n = 28). All elbows were mechanically and histologically tested. The uninjured contralateral elbows of each rat were used as a control group. Capsule release surgery significantly improved (p < 0.01) outcomes 1 week after surgery compared to injury alone and was not significantly different from uninjured elbows. Three weeks after surgery, outcomes worsened, indicating joint stiffening consistent with what is observed clinically. The addition of blebbistatin did not significantly improve outcomes. Future work will investigate relationships among treatment, fibrotic tissue deposition, myofibroblast activity, and biomechanics to determine if blebbistatin is a useful adjunctive therapy for treating joint contracture.

For decades, surgeons have utilized 0.9% normal saline (NS) for joint irrigation to improve visualization during arthroscopic procedures. This continues despite mounting evidence that NS exposure impairs chondrocyte metabolism and compromises articular cartilage function. We hypothesized that chondrocyte oxidative stress induced by low pH is the dominant factor driving NS toxicity, and that buffering NS to increase its pH would mitigate these effects. Effects on chondrocyte viability, reactive oxygen species (ROS) production, and overall metabolic function were assessed. Even brief exposure to NS caused cell death, ROS overproduction, and disruption of glycolysis, pentose phosphate, and tricarboxylic acid (TCA) cycle pathways. NS also stimulated ROS overproduction in synovial cells that could adversely alter the synovial function and subsequently the entire joint health. Buffering NS with 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) significantly increased chondrocyte viability, reduced ROS production, and returned metabolite levels to near control levels while also reducing ROS production in synovial cells. These results confirm that chondrocytes and synoviocytes are vulnerable to insult from the acidic pH of NS and demonstrate that adding a buffering agent to NS averts many of its most harmful effects.

Exosomes have attracted attention due to their ability to promote intercellular communication leading to enhanced cell recruitment, lineage-specific differentiation, and tissue regeneration. The object of this study was to determine the effect of exosomes on cell homing and angiogenic differentiation for pulp regeneration. Exosomes (DPSC-Exos) were isolated from rabbit dental pulp stem cells cultured under a growth (Exo-G) or angiogenic differentiation (Exo-A) condition. The characterization of exosomes was confirmed by nanoparticle tracking analysis and an antibody array. DPSC-Exos significantly promoted cell proliferation and migration when treated with 5 × 10⁸/mL exosomes. In gene expression analysis, DPSC-Exos enhanced the expression of angiogenic markers including vascular endothelial growth factor A (VEGFA), Fms-related tyrosine kinase 1 (FLT1), and platelet and endothelial cell adhesion molecule 1 (PECAM1). Moreover, we identified key exosomal microRNAs in Exo-A for cell homing and angiogenesis. In conclusion, the exosome-based cell homing and angiogenic differentiation strategy has significant therapeutic potential for pulp regeneration.