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Objective To evaluate the short-term safety and effectiveness of amniotic membrane/umbilical cord particulate (AMUC) in managing pain in patients with various severities of knee osteoarthritis (OA). Design Single-center, prospective, investigator-initiated pilot study. Setting Private practice. Subjects A total of 20 knee OA patients aged ≥18 years were enrolled with pain >40 mm, as determined by the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC)–A. Methods Patients received an ultrasound-guided, intra-articular injection of 50 mg of AMUC particulate reconstituted in 2 mL of preservative-free saline. All patients were then monitored at six weeks, 12 weeks, and 24 weeks postinjection. Patients who did not show >30% reduction in pain received a second injection of AMUC at six weeks. WOMAC, Patient Global Assessment, medication usage, and magnetic resonance imaging (MRI) were assessed. Results Knee OA pain significantly decreased from 74.3 ± 17.2 at baseline to 45.0 ± 25.4 at six weeks (P  <   0.01), 35.4 ± 26.6 at 12 weeks (P  <   0.001), and 37.4 ± 26.7 at 24 weeks (P  <   0.001). This pain reduction was associated with a significant improvement in physical function (WOMAC-C) at all time points (P  < 0.05) and stiffness (WOMAC-B) at 12 weeks (P  =   0.01). Eleven patients received a second injection, which was significantly correlated with body mass index >30 kg/m2 (P  =   0.025). MRI evaluation of the overall population revealed an improvement in the severity of bone marrow lesions in seven patients. No adverse events were observed. Conclusions AMUC particulate injection relieved pain and improved physical function in patients with symptomatic knee OA. [ABSTRACT FROM AUTHOR]