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Background: Picosecond-domain laser treatment using a microlens array (MLA) or a diffractive optical element (DOE) generates micro-injury zones in the epidermis and upper dermis. Objective: To investigate interactive tissue reactions between MLA-type picosecond laser pulses and cohesive polydensified matrix hyaluronic acid (CPMHA) filler in the dermis. Methods: In vivo rats with or without CPMHA pretreatment were treated with a 1064-nm picosecond-domain neodymium:yttrium-aluminum-garnet (Nd:YAG) laser using an MLA or DOE. Skin samples were obtained at post-treatment days 1, 10, and 21 and histologically and immunohistochemically analyzed. Results: Picosecond-domain Nd:YAG laser treatment with an MLA-type or a DOEtype handpiece generated fractionated zones of pseudo-cystic cavitation along the lower epidermis and/or upper papillary dermis at Day 1. At Day 21, epidermal thickness, dermal fibroblasts, and collagen fibers had increased. Compared to CPMHAuntreated rats, rats pretreated with CPMHA showed marked increases in fibroblasts and collagen fibers in the papillary dermis. Immunohistochemical staining for the hyaluronic acid receptor CD44 revealed that MLA-type picosecond laser treatment upregulated CD44 expression in the basilar epidermis and dermal fibroblasts. Conclusions: We suggest that the hyaluronic acid-rich environment associated with CPMHA treatment may enhance MLA-type picosecond-domain laser-induced tissue reactions in the epidermis and upper dermis.