MMPs are a group of calcium-dependent zinc-containing enzymes that play a critical role in collagen degradation and matrix remodeling [32].

Matrix metalloproteinase-1 (MMP-1) is a key interstitial collagenase that initiates matrix degradation in damaged skin. Induced by keratinocytes at the wound margins, it binds to type I collagen via the α2β1 integrin. In scar tissue, it primarily targets the initial cleavage of excessively deposited and densely packed type I and type III collagens [31, 32]. Furthermore, MMP-1 attenuates the affinity between type I collagen and α2β1 integrin, facilitating the migration of keratinocytes over type I collagen, thereby promoting re-epithelialization [25, 32, 36]. In a study on photoaged human skin, MMP-1 expression was observed to remain high during the first week post-treatment and decline sharply in the second week [23]. Similarly, another study on mature human burn scars observed elevated MMP-1 expression 48 h postoperatively [31]. This suggests that MMP-1 likely plays a primary role in degrading scar tissue during the early stage following laser treatment.

MMP-3, also known as stromelysin-1, is expressed by keratinocytes in the proximal proliferative population near the leading edge during wound healing [32]. It degrades partially damaged collagen, proteoglycans, elastin, laminin, and fibronectin [32, 36, 37]. In research involving fractional CO2 laser treatment on photoaged human skin, the temporal expression pattern of MMP-3 was observed to be similar to that of MMP-1, remaining at high levels consistently for 7 days post-treatment and dropping sharply in the second week [23]. A similar expression pattern of MMP-3 was also observed in an in vitro 3D skin model, where MMP-3 expression increased on day 1 post-laser but decreased by day 5. The early upregulation of MMP-3 can promote fibroblast-mediated wound contraction and clear the damaged extracellular matrix, providing space for the formation of new tissue; whereas in the later remodeling phase, the downregulation of MMP-3 facilitates the deposition of new tissue [22]. MMP-3 knockdown experiments have shown that its absence leads to reduced keratinocyte proliferation and premature keratinization, ultimately delaying wound closure [37]. These findings underscore the role of MMP-3 in wound healing after therapy.

MMP-9 (also known as gelatinase B) is primarily responsible for degrading gelatin produced after initial cleavage by interstitial collagenases. It also possesses the ability to degrade various matrix components, including type IV and type VII collagen, elastin, and fibrillin [32]. This broad substrate degradation capability enables it to effectively clear matrix debris and relieve the physical constraints of the basement membrane on keratinocytes, thereby promoting epithelial regeneration and dermal remodeling. On day 1 after fractional CO2 laser treatment of photoaged human skin, MMP-9 immunostaining was observed, primarily reflecting neutrophil infiltration. This is because early MMP-9 is mainly stored in neutrophil granules and is rapidly released upon infiltration. However, MMP-9 gene expression showed a delayed increase, maintaining high levels for 1–3 weeks post-treatment [23]. In a study using a red Duroc pig model of hypertrophic scarring, immunofluorescence results similarly observed a mild elevation in MMP-9 levels on day 35 post-laser treatment [38]. Compared to MMP-1 and MMP-3, MMP-9 exhibits a more sustained expression, allowing it to clear collagen fragments generated by previous matrix proteinase degradation, thereby preparing for subsequent tissue repair.

MMP-13 is also a potent, broad-spectrum collagenase capable of degrading almost all types of fibrillar collagen, and it can coordinate with MMP-9 in the re-epithelialization process [35, 37]. Following fractional CO2 laser treatment, MMP-13 expression often exhibits a relatively delayed characteristic. For example, in post-treatment photoaged human skin, MMP-13 was observed to peak on day 14 [39]. This suggests that it may be more involved in the later stages of clearing residual collagen debris and orderly rearranging newly synthesized collagen. However, in another study on mature human burn scars, no statistically significant difference in MMP-13 expression levels was observed 48 h post-treatment compared to pre-treatment levels [31]. This could be due to the detection time preceding the MMP-13 expression peak, but it also implies that MMP-13 might not play a core role during fractional CO2 laser treatment of hypertrophic scars.

MMP-12 (also known as macrophage metalloelastase) is primarily responsible for the specific degradation of elastin, while also possessing the capability to degrade matrix components [32]. In 3D skin models, a significant downregulation of MMP-12 can be observed on day 5 post-treatment [22]. Another study on photoaged human skin similarly observed that MMP-12 was immediately downregulated after fractional CO2 laser treatment and remained in a downregulated state for an extended period [39]. This sustained low-expression state may suggest a delicate “selective remodeling” mechanism in fractional laser therapy: while other MMPs actively degrade old collagen, the tissue specifically protects the elastic fiber network from excessive destruction by downregulating MMP-12. This mechanism also provides a plausible molecular biological explanation for the clinically observed enhancement of the elastic fiber network in scar tissues following treatment [35].

Following fractional CO2 laser treatment, the expression of HSP family members is upregulated, which aids in cellular resistance to laser-induced damage and promotes tissue repair and wound healing [12].

HSP70 is a critical marker of thermal injury. Functioning as a molecular chaperone, it assists cells in handling abnormally folded proteins, prevents protein misfolding and aggregation, plays a vital role in cellular repair and wound healing, and can induce the expression of multiple growth factors such as TGF-β [12, 36]. A study using a skin explant model showed that HSP70 was significantly upregulated 1 h after fractional CO2 laser treatment, peaked between 1 and 24 h, and then significantly declined within 7 days, with no notable differences across different energy settings [36]. In contrast, a study on normal human skin observed enhanced HSP70 staining starting at 4 h post-treatment, which remained at high levels for up to 3 months postoperatively, indicating a persistent molecular-level healing response [28]. These two distinct temporal patterns may stem from differences in the research models.

HSP47 is an endoplasmic reticulum (ER)-resident chaperone specific to collagen synthesis. It interacts with procollagen in the ER, assisting in the folding and assembly of procollagen α1(I) and α2(I) chains, thereby promoting the maturation and stable deposition of newly formed collagen. A deficiency of HSP47 impairs the maturation of type I and type IV collagen, affecting the formation of microfibrils and the basement membrane. Studies indicate that HSP47 expression in the dermis is positively correlated with the rate of collagen formation. In a study by Helbig et al. on photo-damaged human skin, a slight increase in the distribution and intensity of HSP47 was observed within 14 days across different energy groups, peaking at 3–14 days post-treatment with no significant differences among the energy groups [12]. Conversely, in another study on normal human skin, immunohistochemical results indicated that HSP47 expression increased on the fifth postoperative day and remained at a high level for at least 3 months [28]. This suggests that HSP47 is involved in long-term wound healing and collagen remodeling. Although existing data are mostly derived from non-scarred tissue, given the core regulatory role of HSP47 in collagen maturation, it is highly likely to play a critical role in guiding the orderly rearrangement of nascent collagen and determining the ultimate quality of dermal matrix remodeling within the microenvironment of fractional laser-remodeled pathological scars.

TGF-β is a crucial cytokine that regulates cell proliferation, migration and matrix remodeling throughout the process of cutaneous wound healing. It is primarily released by degranulated platelets and macrophages at the site of injury and influences multiple phases of skin repair [31]. During the inflammatory phase, TGF-β recruits macrophages and granulocytes via chemotaxis and mediates the release of proinflammatory cytokines, including interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) [20]. In the proliferative phase, it induces the expression of migration integrins in keratinocytes, promoting their migration toward the wound edge and thus accelerating re-epithelialization. During matrix formation and remodeling, TGF-β exhibits strong pro-fibrotic characteristics; it not only attracts and activates fibroblasts, inducing their differentiation into myofibroblasts with high α-SMA expression to stimulate collagen synthesis, but it also downregulates the expression of matrix metalloproteinases (MMPs) to inhibit collagen degradation. Consequently, the abnormal hyperactivity of the TGF-β signaling pathway is considered a key driver in the formation of pathological scars [4, 12, 20, 29, 36, 40, 41]. A study using a single fractional CO2 laser treatment at 2.77 J/cm² on photoaged human skin demonstrated that TGF-β expression peaked 3 days post-treatment and then gradually declined until day 30 [20]. A similar biphasic, time-dependent response of initial upregulation followed by downregulation was also observed in a normal mouse skin model [21]. This early and transient elevation helps initiate tissue repair, while the subsequent timely decline relieves the inhibitory effect on MMPs, preventing the occurrence of excessive fibrosis.

The TGF-β family comprises three primary isoforms: TGF-β1, TGF-β2, and TGF-β3. Traditionally, TGF-β1 and TGF-β2 are believed to exhibit pro-fibrotic properties, whereas TGF-β3 possesses anti-fibrotic effects [31]. Among them, TGF-β1 is highly expressed in hypertrophic scars and plays a crucial role in their pathogenesis [29, 40]. In a study by Qu et al. on mature human burn scars, the expression of TGF-β1 showed no significant change 48 h post-laser irradiation. However, at this time point, the expressions of TGF-β2 and TGF-β3 were significantly downregulated, suggesting that within the microenvironment of mature burn scars, TGF-β3 may similarly exert a pro-fibrotic effect [31]. In a study by Makboul et al., immunohistochemical results revealed a marked decrease in TGF-β1 in human hypertrophic scar tissue 6 months after 4 sessions of fractional CO₂ laser treatment [29], a result similarly observed in another study on human scar tissues [35]. The downregulation of TGF-β1 inhibits the sustained activation of myofibroblasts, which also explains the subsequent downregulation of α-SMA expression in the treated scar tissue. Thus, it is evident that fractional CO2 laser therapy may participate in scar resolution by downregulating TGF-β2 and TGF-β3 in the early stages and downregulating TGF-β1 in the later stages.

Recent studies have demonstrated that miRNAs also play a pivotal role in skin healing and scar remodeling induced by fractional CO2 laser. In hypertrophic scar tissues, 152 miRNAs are differentially expressed, suggesting their active involvement in the regulation of fibrosis [42]. Furthermore, the miR-17–92 cluster can participate in the regulation of the fibrotic process by targeting various regulatory components within the TGF-β signaling pathway; notably, miR-18a exerts its effects by inhibiting the expression of Smad4 [31, 43]. The miR-17–92 cluster can also downregulate anti-angiogenic factors to promote angiogenesis, while miR-19a/b and miR-20 serve as inflammatory modulators during wound healing, effectively suppressing keratinocyte inflammation and promoting wound closure [43,44,45]. Following fractional CO2 laser treatment, expressions of miR-18a and miR-19a within the miR-17–92 cluster are significantly upregulated in human hypertrophic scar tissues [31]. These findings suggest that fractional CO2 laser may selectively activate specific miRNAs to negatively regulate TGF-β signaling and inhibit local inflammation, thereby facilitating scar remodeling.

bFGF plays a critical multi-phase role in the human wound healing process. As a broad-spectrum mitogen, bFGF can promote the growth and differentiation of various cell types, exhibit potent angiogenic and mitogenic activities, and participate in the entire process of the inflammatory phase, proliferative phase and remodeling phase [20, 31, 41, 46, 47]. A study utilizing a 2.77 J/cm² fractional CO2 laser on photoaged human skin observed a gradual upregulation of bFGF expression levels over 30 days post-treatment; however, this trend was absent in the 2.07 J/cm² and 4.15 J/cm² treatment groups [20]. Conversely, in another study treating ten patients with mature burn scars, a significant decrease in endogenous bFGF expression was observed 48 h post-treatment [31]. These discrepancies in expression dynamics may be attributed to differences in baseline skin pathological states, observation time, and fluence settings.

During the early inflammatory phase of wound healing, bFGF can recruit leukocytes to the wound site to initiate healing. During the proliferative phase, it enhances the migration and proliferation of fibroblasts and keratinocytes, promotes re-epithelialization, stimulates balanced synthesis of ECM such as collagen and hyaluronic acid, induces angiogenesis to improve local oxygenation and nutrition and thus accelerates tissue regeneration. During remodeling, bFGF upregulates MMP-1 expression in HS-derived fibroblasts and inhibits the differentiation of myofibroblasts derived from fibroblasts and ESCs (epidermal stem cells). This mechanism prevents excessive collagen deposition and fibrosis, ultimately averting the formation of pathological scars [20, 31, 46, 48,49,50].

Given that fractional laser treatment of mature scars may lead to the early downregulation of endogenous bFGF expression, the clinical strategy of exogenous supplementation is essential. Clinical studies have confirmed that fractional CO₂ laser combined with rb-bFGF gel yields superior therapeutic efficacy in treating mature facial burn scars compared to laser monotherapy. The timely intervention of exogenous bFGF not only accelerates tissue regeneration and the healing process but also significantly reduces the incidence of postoperative complications, such as persistent erythema and PIH [46, 48].