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Samples were withdrawn after 0, 2, 4, 24, 48 and 72 hours and incubated with enzyme and detection reagent for 10 minutes before detection using a SpectraMax i3 plate reader at 550 nm excitation and 595 nm emission

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The mechanisms most relevant to knee pain: VEGF upregulation stimulates new blood vessel formation in cartilage and connective tissue, areas that are notoriously slow to heal due to limited blood supply Nitric oxide modulation regulates inflammation and promotes vasodilation, improving blood flow to damaged tissue Collagen synthesis accelerates organized collagen deposition at repair sites instead of disorganized scar tissue Tendon-to-bone healing particularly relevant at the patella tendon and other knee attachment points Animal studies across rat, rabbit, and canine models showed consistent findings across these pathways