CO2 Laser for Esthetic Healing of Injuries and Surgical Wounds with Small Parenchymal Defects in Oral Soft Tissues

A number of studies have recently demonstrated the effectiveness of CO2 laser irradiation for the repair and regeneration of scar tissue from injuries or surgical wounds. However, such studies of the oral mucosa are highly limited. Previous studies using CO2 laser irradiation have indicated that two factors contribute to esthetic healing, namely, artificial scabs, which are a coagulated and carbonized blood layer formed on the wound surface, and photobiomodulation therapy (PBMT) for suppressing wound scarring and promoting wound healing. This review outlines basic research and clinical studies of esthetic healing with the use of a CO2 laser for both artificial scab formation by high-intensity laser therapy and PBMT in the treatment of injuries and surgical wounds with small parenchymal defects in oral soft tissues. The results showed that the wound surface was covered by an artificial scab, enabling the accumulation of blood and the perfusion necessary for tissue regeneration and repair. Subsequent PBMT also downregulated the expression of transformation growth factor-b1, which is involved in tissue scarring, and decreased the appearance of myofibroblasts. Taken together, artificial scabs and PBMT using CO2 lasers contribute to the suppression of scarring in the tissue repair process, leading to favorable esthetic and functional outcomes of wound healing.


Introduction
During the healing of injuries and surgical wounds with small parenchymal defects in oral soft tissues, scarring (e.g., formation of hypertrophic scars, contracture and epithelial concavity) sometimes occurs, affecting esthetic and functional outcomes.Scarring and associated problems can also occur due to suturing performed to achieve early wound closure and prevent wound infections [1,2] and due to the use of a wound dressing [3,4].
We have been studying CO 2 laser treatment of tooth extraction sockets and reported basic research results such as the promotion of wound healing, preservation of the alveolar crest and suppression of mucosal scarring around the extraction sockets [25][26][27][28].We also reported clinical research results on CO 2 laser treatment that suppressed scarring during the healing of open lip vermillion wounds caused by injuries, resulting in esthetic tissue regeneration [29].Two factors contribute to the esthetic wound healing achieved by CO 2 laser treatment.One is the presence of "artificial scabs" formed by coagulation and carbonization of the surface of blood accumulated in injuries and surgical wounds by high-intensity laser therapy (HILT).The other is photobiomodulation therapy (PBMT), which is expected to promote wound healing and suppress scar formation.
This review outlines basic research and clinical studies on esthetic healing by use of a CO 2 laser for both artificial scab formation by HILT and PBMT in the treatment of injuries and surgical wounds with small parenchymal defects in oral soft tissues and provides key points of treatment procedures based on the findings from some clinical case examples.
First, we briefly explain scar formation in primary healing and secondary healing.

Wound Healing and Scarring
The presence of myofibroblasts is essential for the closure of injuries and surgical wounds.Myofibroblasts, which have smooth muscle cell-like contractile activity, are differentiated from fibroblasts in granulation tissue upon expression of TGF-b1.Then, scarring and scar contracture occur, and early wound closure is promoted by total coverage of wounds by epithelia [30][31][32][33].
However, this healing mechanism can cause unfavorable esthetic and functional outcomes because of the development of hypertrophic scars, scar contracture and epithelial concavity on the wound surface [34][35][36].The degree of scar formation varies depending on the size, area and depth of parenchymal defects.

Primary Healing and Scar Formation
Primary wound healing occurs when an incision made by a scalpel is closed by tight suturing of the dermal edges.In principle, primary healing can be achieved when wounds are not contaminated and do not require debridement.However, it is possible after thorough debridement if the wound is closed by suturing the dermal edges without leaving dead space.Also, when suturing, it is important to carefully align the epithelium on one dermal edge with that of the other and the submucosa on one dermal edge with that of the other.
The following problems are associated with primary healing: (1) a linear scar forms at the surface plane of the closed wound edges because of the scar tissue involved and contraction of the wound surface; (2) when suturing or reefing of the muscle layers is performed, the wound surface and surrounding tissue are pulled, leading to motor dysfunction due to contracture and tension in the future; and (3) when suturing or reefing of the wound edges is not tight enough, dead space forms subcutaneously or at the deep position beneath the wound, and then granulation tissue forms to fill the dead space, which is eventually replaced by scar tissue.

Secondary Healing and Scar Formation
Secondary healing occurs in open wounds that lack epithelia.After thorough debridement, blood on the wound surface coagulates, and defects are filled by the granulation tissue formed via the deposition of fibroblasts and angiogenesis.Then, fibroblasts differentiate into myofibroblasts in granulation tissue, and the granulation tissue is replaced by scar tissue, resulting in repair and regeneration.At the same time, the epithelium around the wound extends to cover the entire area of the wound, leading to healing.
Recently, to prevent scar formation like that described above, dressing therapy, where the wound is covered by a dressing, has been used for open wounds [3,4].This facilitates early wound healing by keeping the wound surface adequately moist, or more precisely, by keeping blood and effusion containing cytokines that induce migration, division and proliferation of cells within the wound, and by protecting regenerated immature epithelia and granulation tissue from contamination and infection (Figure 1a).However, the following problems are associated with this treatment: (1) there is a risk of aggravation of infection in the closed environment (main causes are the presence of bacteria, inadequate debridement, oxygen deficiency); (2) epithelial healing may be inadequate due to the accumulation of excessive effusion in the highly closed conditions; (3) there is a risk of damage to the wound surface upon removal of the wound dressing; and (4) fixation of a wound dressing over highly mobile tissue is difficult, causing misalignment or opening of wound edges, which may result in dead space formation and consequent formation of a large amount of scar tissue.
Recently, to prevent scar formation like that described above, dressing therapy, where the wound is covered by a dressing, has been used for open wounds [3,4].This facilitates early wound healing by keeping the wound surface adequately moist, or more precisely, by keeping blood and effusion containing cytokines that induce migration, division and proliferation of cells within the wound, and by protecting regenerated immature epithelia and granulation tissue from contamination and infection (Figure 1a).However, the following problems are associated with this treatment: (1) there is a risk of aggravation of infection in the closed environment (main causes are the presence of bacteria, inadequate debridement, oxygen deficiency); (2) epithelial healing may be inadequate due to the accumulation of excessive effusion in the highly closed conditions; (3) there is a risk of damage to the wound surface upon removal of the wound dressing; and (4) fixation of a wound dressing over highly mobile tissue is difficult, causing misalignment or opening of wound edges, which may result in dead space formation and consequent formation of a large amount of scar tissue.Next, we take into account the characteristics of CO2 lasers and explain to what extent the CO2 laser treatment can suppress scar formation in primary healing and secondary healing of injuries and surgical wounds with small parenchymal defects in oral soft tissues.

Characteristics of CO2 Laser
Dental CO2 lasers emit long-wavelength light (10.6 mm) that is strongly absorbed by water.Therefore, upon irradiation of the skin, the thermal energy of a CO2 laser is mostly absorbed before reaching a depth of 0.05 mm beneath the skin surface, causing no impact on the deep tissue [37][38][39].Similarly, in the irradiation of blood, coagulation and carbonization occur only at the surface, and blood under the surface layer is unaffected.
However, excessive CO2 laser irradiation due to inappropriate conditions (e.g., output, irradiation mode, irradiation time, irradiation distance) and inappropriate maneuvers can cause irreversible changes and delayed healing.

Secondary Healing-like Effect Using a CO2 Laser
The two factors of artificial scabs and PBMT are involved in achieving secondary healing using a CO2 laser.We explain these factors below.

Presence of Artificial Scabs
Artificial scabs, formed by coagulation and carbonization of blood at the wound surface by HILT, are necessary for the repair and regeneration of parenchymal defects associated with injuries and surgical wounds to obtain the original tissue form [25][26][27][28][29]. These artificial scabs play a role similar to that of wound dressings in dressing therapy (Figure Next, we take into account the characteristics of CO 2 lasers and explain to what extent the CO 2 laser treatment can suppress scar formation in primary healing and secondary healing of injuries and surgical wounds with small parenchymal defects in oral soft tissues.

Characteristics of CO 2 Laser
Dental CO 2 lasers emit long-wavelength light (10.6 mm) that is strongly absorbed by water.Therefore, upon irradiation of the skin, the thermal energy of a CO 2 laser is mostly absorbed before reaching a depth of 0.05 mm beneath the skin surface, causing no impact on the deep tissue [37][38][39].Similarly, in the irradiation of blood, coagulation and carbonization occur only at the surface, and blood under the surface layer is unaffected.
However, excessive CO 2 laser irradiation due to inappropriate conditions (e.g., output, irradiation mode, irradiation time, irradiation distance) and inappropriate maneuvers can cause irreversible changes and delayed healing.

Secondary Healing-like Effect Using a CO 2 Laser
The two factors of artificial scabs and PBMT are involved in achieving secondary healing using a CO 2 laser.We explain these factors below.

Presence of Artificial Scabs
Artificial scabs, formed by coagulation and carbonization of blood at the wound surface by HILT, are necessary for the repair and regeneration of parenchymal defects associated with injuries and surgical wounds to obtain the original tissue form [25][26][27][28][29]. These artificial scabs play a role similar to that of wound dressings in dressing therapy (Figure 1b).They have a "space-making effect" to retain effusion and blood required for repair and regeneration of tissue to its original form, preserve the moist condition for moist wound healing and protect immature epithelial and granulation tissues from contamination and infection, thereby facilitating early wound healing.Problems associated with wound dressings, explained in Section 2.2, are absent in the case of artificial scabs: problems (1) and ( 2) are unlikely because artificial scabs are breathable; problem (3) is unlikely because they fall off naturally rather than requiring forced removal; and problem ( 4) is unlikely because laser soldering of artificial scabs formed over the mobile tissue to the surrounding tissues reduces the risk of peeling [40,41].

Preventive Effect of PBMT on Scarring
PBMT is expected to promote wound healing and prevent scar formation.This review focuses on scar prevention by PBMT in the healing of oral soft tissue.Those who are interested in the promotion of healing by PBMT are encouraged to refer to our previous studies [25][26][27].PBMT inhibits differentiation of fibroblasts to myofibroblasts in granulation tissue and inhibits or improves scarring.
Recently, the effectiveness of PBMT using a CO 2 laser for inhibiting or improving scar formation associated with trauma, burns, surgical wounds and acne in skin tissue was demonstrated in clinical studies using a biopsy or a scar scale.Pathohistological and biochemical examination of wound biopsy samples showed inhibition of collagen fiber production [5,6,9], normalization of collagen fiber orientation [5], normal regeneration of and increases in dermal collagen with elastic fibers [8,10], thinning in the stratum corneum [5,9] and downregulation of expression of TGF-b1 involved in differentiation of fibroblasts into myofibroblasts [5,7,9].With respect to appearance, studies of scar healing, such as those using the Vancouver Scar Scale or ultrasound measurement, have demonstrated improvements in the hardness [10,14], thickness [11,16] and flexibility and elasticity [10,14] of scars.In studies of motor function at scar sites [12], studies using the Patient Scar Assessment Questionnaire showed improvements in appearance and scar awareness [10][11][12][13][14][15][16].
Basic studies using experimental animals with artificially formed scar tissue in skin tissue have shown that scar formation can be alleviated by PBMT through downregulation of TGF-b1 expression [17,20,21], which decreases expression of the myofibroblast marker α-smooth muscle actin (α-SMA) [21,22], decreases the amount and density of excessive collagen fibers produced in granulation tissue [17][18][19][20][21], decreases the disruption of fiber orientation [19,20], decreases the scar elevation index [21], decreases the micro-vessel density [18] and thins the fiber layer [20].Also, PBMT induces downregulation of fibroblast expression in scar tissue [18,19,21], where basic fibroblast growth factor (bFGF) plays a role.During the maturation phase of wound healing, a scar forms when many fibroblasts are present in the tissue.Secretion of bFGF induces apoptosis of fibroblasts, thereby inhibiting and reducing scar tissue formation due to the production of excessive collagen fibers.On the other hand, during the inflammatory and proliferative phases, bFGF promotes cell division to increase fibroblasts, thereby promoting repair and regeneration of the wound.During these phases, PBMT stimulates bFGF secretion to promote repair and regeneration of normal tissue without scarring [23,24].
The relationship between PBMT and TGF-b1 has been studied with respect to suppression of scarring and promotion of wound healing.Laser beams affect mitochondrial cytochrome-C oxidase, thereby influencing the production of adenosine triphosphate (ATP), which is a major source of energy for cell functions.These responses include the generation of reactive oxygen species (ROS) that activate nuclear factor-κB, which plays a role in the signaling cascade, including wound shrinkage, fibroblast differentiation and collagen production.ROS induces extracellular activation of TGF-b1.Also, downregulation of TGF-b1 signaling enhances the formation of keloids and hypertrophic scars.However, PBMT increases ATP production, but the level of ROS remains low.As a result, the downregulation of TGF-b1 by PBMT affects the reduction and inhibition of profibrotic gene synthesis and collagen synthesis.These are likely to indicate the suppression of wound tissue scarring [42,43].On the other hand, TGF-b1 is a potent regulator of inflammatory responses and is usually upregulated in the early phase of wound healing.However, when comparing treatment with and without PBMT, we found no significant differences in TGF-b1 expression in the inflammatory phase but a significant decrease by PBMT in the proliferative phase of wound healing, indicating a suppressive effect on wound tissue scarring [26,28].
After PBMT, TGF-b2, like TGF-b1, is also involved in recruiting fibroblasts and immune cells from the circulation and wound edges to the wounded area, thereby promoting granulation tissue formation and collagen synthesis [44].On the other hand, a different study showed the appearance of apoptotic epithelial cells and fibroblasts after PBMT [45].This phenomenon is likely to show suppression of tissue scarring through a reduction in excessive collagen production associated with wound healing.In contrast to TGF-b1 and TGF-b2, TGF-b3 possibly reduces scarring in adults and promotes scarless healing in fetuses, but no study has provided evidence on TGF-b3 in relation to PBMT.Basic studies on PBMT using a CO 2 laser and TGF-b are limited, and future studies are awaited.
Taken together, both clinical and basic studies have demonstrated that PBMT is effective in suppressing scar tissue formation.At present, although suppression of scar formation has been verified, complete healing is difficult solely with PBMT using a CO 2 laser.To address this, combination with a laser of a different wavelength or an agent has been studied to improve the reliability of treatment effectiveness [7,9,[17][18][19][20][21].
As described above, studies of scars in the oral mucosa, including our studies, are limited compared with those of scars in skin tissue.This can be explained by the rarity of scar formation in the oral mucosa due to differences in turnover (a few days to 2 weeks for the oral mucosa vs. approximately 4 weeks for skin tissue).However, depending on the treatment procedures used, mucosal concavity and recession occur, influencing the esthetic outcomes and motor functions of the tongue, lips and cheeks.Also, it is important to understand that the lips are complex tissues with a transition between oral mucosa and skin tissue.
Next, we describe secondary healing achieved in clinical cases by treatment procedures using a CO 2 laser (Figure 2).Funakoshi.Consent was obtained from the patients in all cases.Reproduced from [29] under Creative Commons CC BY-NC 4.0.

Treatment of the Lips Using a CO 2 Laser
Achieving favorable esthetic outcomes is crucial in the facial area, including the lips, so careful postoperative treatment is important.

Mucocele of the Lip
A mucocele of the lip is thought to be caused by impaired outflow of mucus from a minor salivary gland due to damage to the opening of the minor salivary gland duct present under the labial mucosa.
In conventional mucocele removal, infiltration anesthesia is applied to the area around the mucocele, and an incision is made on the normal mucosa without damaging the mucocele using a scalpel.Then, the lesion is fully enucleated from the surrounding tissue using a surgical scalpel, mucosal elevator or mosquito forceps, and lastly, suturing is performed (Figure 3a-e) [46].
On the other hand, in mucocele removal using a CO 2 laser, tissues are cut and vaporized by a CO 2 laser instead of being cut by a scalpel, and basically, no suturing is required.The most important point of this method is to retain blood in the space generated after mucocele removal and along the lip morphology and to form an artificial scab on the surface.Then, artificial scabs are strongly soldered to the surrounding tissues to avoid detachment [40,41], and PBMT is subsequently applied several times before the end of the procedure (Figures 2a-c and 3a,f-k).This method frees surgeons from suturing and also frees patients from pain associated with suturing and uncomfortable symptoms such as tension and contracture of the lip.The surgical procedure for hemangioma resection, although we have not performed it, is basically the same as that described above [47].
Recently, clinical studies have shown that this treatment procedure can reduce bleeding and pain, requires a short time and is associated with almost no or no recurrence [48,49].However, findings regarding esthetic and motor function outcomes associated with scar formation in treated tissue have not yet been reported in detail.
Clinical studies using lasers of other wavelengths have also been reported [50,51], but no studies using biopsy have yet been reported.Also, there are specific problems associated with the use of lasers.For example, when a laser tip is in direct contact with the skin for vaporization and cutting in treatment using a laser that penetrates tissue (e.g., a diode laser), bleeding during the treatment is reduced, but heating of the surrounding tissue induces protein coagulation, which is associated with a risk of delayed wound healing and some scar formation [52,53].Compared with CO 2 lasers, when Er.YAG (erbium-yttrium-aluminumgarnet) lasers, which are absorbed at the surface, are used, more bleeding is expected, albeit without the effect of heating the surrounding tissue during treatment [39,52,54].On the other hand, in mucocele removal using a CO2 laser, tissues are cut and vapo ized by a CO2 laser instead of being cut by a scalpel, and basically, no suturing is require The most important point of this method is to retain blood in the space generated aft mucocele removal and along the lip morphology and to form an artificial scab on the su face.Then, artificial scabs are strongly soldered to the surrounding tissues to avoid d tachment [40,41], and PBMT is subsequently applied several times before the end of t procedure (Figures 2a-c and 3a,f-k).This method frees surgeons from suturing and al frees patients from pain associated with suturing and uncomfortable symptoms such tension and contracture of the lip.The surgical procedure for hemangioma resection, a hough we have not performed it, is basically the same as that described above [47].

Open Lip Vermillion Wounds
The lips are the part of the face most prone to injury.Treatment of open lip vermillion wounds includes thorough debridement of contaminated tissue at the injured site and then advanced suturing (e.g., V-Y advancement flap [1,2]) that achieves esthetic and functional reconstruction of the lips.Although debridement is necessary, it must be kept to a minimum when treating tissues (e.g., the face) where favorable esthetic outcomes are particularly important.It is highly likely that forceful reefing results in scar formation along the wound edges that are closed and consequent interruption of the vermillion border (Figure 4a-e).Similar findings were shown in suturing in the surgery of the cleft lip [55,56].Also, because the lips are highly mobile, wound dehiscence and the formation of dead space in the deep part beneath the wound may occur, as described in problem (4) of Section 2.2.
minimum when treating tissues (e.g., the face) where favorable esthetic outcomes are particularly important.It is highly likely that forceful reefing results in scar formation along the wound edges that are closed and consequent interruption of the vermillion border (Figure 4a-e).Similar findings were shown in suturing in the surgery of the cleft lip [55,56].Also, because the lips are highly mobile, wound dehiscence and the formation of dead space in the deep part beneath the wound may occur, as described in problem (4) of Section 2.2.On the other hand, in treatment using a CO2 laser, debridement and artificial scab formation are accomplished.In this procedure, after minimum vaporization of On the other hand, in treatment using a CO 2 laser, debridement and artificial scab formation are accomplished.In this procedure, after minimum vaporization of contaminated tissue by the laser, blood is allowed to accumulate in the dead space formed at the site of the tissue defect and along the lip morphology, and artificial scabs are formed on the blood surface and strongly soldered to the surrounding tissues [40,41].Then, PBMT is performed several times (Figures 2d-f and 4a-c,f-k).It should be noted that the formation of artificial scabs without careful consideration results in irregular closure of the lip wound edges, potentially causing concavity, unevenness and hypertrophic scar formation.Thus, it is important to align and close the lip wound edges by light suturing and then to form artificial scabs [29].

Treatment Using a CO 2 Laser after Tooth Extraction
The guidelines for use of dental lasers formulated by the U.S. Food and Drug Administration recommend the "coagulation of extraction sites" using a CO 2 laser.
In the tooth extraction procedure using a CO 2 laser, tooth extraction is performed in the conventional manner, but hemostasis is achieved by laser coagulation.Suturing or compression are mainly used for hemostasis in tooth extraction, but wound closure by suturing causes excessive tension in the surrounding mucosa and a consequent reduction in the height of the alveolar mucosa, and the gauze pieces and dental cotton rolls used for compression hemostasis absorb blood.Thus, with these conventional hemostasis procedures, an adequate amount of blood, which is necessary for alveolar bone regeneration in the extraction socket, cannot be secured in the extraction socket, resulting in vertical alveolar bone resorption and a mucosal concavity at the site of the extraction wound (Figures 5a,b and 6a-e).
In the tooth extraction procedure using a CO2 laser, tooth extraction is performed in the conventional manner, but hemostasis is achieved by laser coagulation.Suturing or compression are mainly used for hemostasis in tooth extraction, but wound closure by suturing causes excessive tension in the surrounding mucosa and a consequent reduction in the height of the alveolar mucosa, and the gauze pieces and dental cotton rolls used for compression hemostasis absorb blood.Thus, with these conventional hemostasis procedures, an adequate amount of blood, which is necessary for alveolar bone regeneration in the extraction socket, cannot be secured in the extraction socket, resulting in vertical alveolar bone resorption and a mucosal concavity at the site of the extraction wound (Figures 5a,b and 6a-e).On the other hand, in the treatment using a CO2 laser, an artificial scab is formed after letting blood accumulate to the height of the surrounding mucosa of the extraction socket, and it is soldered to the surrounding mucosa [40,41], and PBMT is then performed several times (Figures 2g-i, 5c and 6f-j).On the other hand, in the treatment using a CO 2 laser, an artificial scab is formed after letting blood accumulate to the height of the surrounding mucosa of the extraction socket, and it is soldered to the surrounding mucosa [40,41], and PBMT is then performed several times (Figures 2g-i, 5c and 6f-j).
These scabs prevent the extension and invagination of the surrounding mucosa into the extraction socket, thereby playing a role in space-making for bone regeneration within the extraction socket.Subsequent PBMT is also important for treating extraction wounds.It suppresses the contracture of a scar developed on the mucosal epithelium through marked downregulation of TGF-b1 and α-SMA in the mucosal epithelium of extraction wounds, as shown in our previous studies [26,28] and described in Section 4.2.PBMT also plays a role in the abovementioned space-making.In rats, we also demonstrated that activation of bone remodeling and formation of new bone with a cross-linking pattern occurred in the shallow layer of an extraction socket at the depth of the CO 2 laser light penetration.Such new bone with a cross-linking pattern serves as a bone lining under the mucosa of the extraction wound, thereby preventing extension of the mucosal epithelium into the extraction socket and making space for bone regeneration [25][26][27].
Taken together, artificial scabs and PBMT contribute to preserving alveolar bone height to the greatest extent possible and also to preventing the formation of a mucosal epithelial concavity at the site of the extraction wound.
Note that when marked alveolar bone resorption is present due to periodontal diseases or other conditions, an adequate height of the extraction socket wall, which is a part of the alveolar bone supporting a dentition, cannot be secured.Consequently, an adequate amount of blood cannot be accumulated.This results in a reduction in the regenerated bone height, causing mucosal concavity at the site of the extraction wound.

Free Gingival Grafting Using a CO 2 Laser
In this treatment, for cases with no or little attached gingiva, a gingival flap harvested from a donor site (e.g., part of the palatal mucosa) is transplanted to a recipient site to increase the width of the attached gingiva or to cover a root with gingival recession.A gingival flap is placed firmly in the recipient site to avoid the formation of dead space and is then sutured to the surrounding mucosa.Currently, the main procedure for exposed donor sites is covering them with a periodontal pack or artificial dermis.
On the other hand, a CO 2 laser can be used in this treatment to form artificial scabs on the surface of blood accumulated at the donor site.These scabs, like a periodontal pack or artificial dermis, are likely to play a role similar to that of a wound dressing (see Section 4.1).A CO 2 laser can also be used after suturing for soldering wound edges in the spaces between suture points [40,41]; the treatment is completed after performing PBMT several times (Figures 2j-l and 7).We use a scalpel, but not a CO 2 laser, when harvesting a gingival flap to avoid a risk of protein coagulation by heating due to laser irradiation at the margins of the flap edges.
Studies of CO 2 lasers in this treatment are very limited.In one study, gingival flaps of good quality without thermal protein denaturation were harvested, and high root coverage and significant gains in keratinized gingiva were achieved [57].In another study, recession of the gingival flap 1 year after grafting was significantly decreased by postgraft irradiation of the recipient site [58].However, basic research investigating whether CO 2 lasers promote the healing of mucosal epithelial wounds and whether they influence scar formation at donor and recipient sites has not yet been reported.
PBMT using a laser that penetrates tissue as an adjunct to wound repair markedly accelerated re-epithelization and contributed to wound pain relief [59].It was noted that PBMT using a Nd:YAG laser increased the expression of TGF-b1 at the donor site to stimulate fibroblasts to close the wound in the early phase of wound healing, but the TGF-b1 expression was decreased in the late phase [60].As described in Section 4.2, PBMT may work to avoid scar formation as wound healing progresses to the maturation phase.

Figure 1 .
Figure 1.Dressing therapy with wound dressing and wound covering with an artificial scab formed using a CO2 laser: (a) wound dressing; (b) artificial scab.

Figure 1 .
Figure 1.Dressing therapy with wound dressing and wound covering with an artificial scab formed using a CO 2 laser: (a) wound dressing; (b) artificial scab.

16 Figure 2 .
Figure 2. Cases and surgical methods with artificial scabs formed using a CO2 laser: (a-c) removal of a mucocele; (d-f) treatment of open lip vermillion wounds; (g-i) treatment after tooth extraction; (j-l) treatment of the donor site for a free gingival graft; (a) preoperative; (b) after removal of the mucocele; (c) an artificial scab formed on the surface of the resection wound; (d) immediately after open lip vermillion wounds; (e) artificial scab formed in open lip vermillion wound; (f) 6 months after open lip vermillion wound; (g) the extraction socket fully filled with blood immediately after tooth extraction; (h) an artificial scab formed on the surface of blood filled in the extraction socket; (i) day 2 after tooth extraction; (j) preoperative; (k) an artificial scab formed after the donor site of a gingival flap filled with blood clots; (l) 1 month after surgery.Photos by (d-i) co-author Dr. H. Fu-

Figure 2 .
Figure 2. Cases and surgical methods with artificial scabs formed using a CO 2 laser: (a-c) removal of a mucocele; (d-f) treatment of open lip vermillion wounds; (g-i) treatment after tooth extraction; (j-l) treatment of the donor site for a free gingival graft; (a) preoperative; (b) after removal of the mucocele;

Figure 3 .
Figure 3. Removal of mucocele using a CO2 laser: (a) before removal of mucocele; (b-e) convention surgical method; (f-k) surgical method using CO2 laser; (b) designing a spindle incision line ov the mucocele along a lip wrinkle and making a submucosal incision; (c) removing a mucocele wit out damaging it; (d) hemostatic suturing; (e) high likelihood of formation of a linear scar and mucosal concavity along the sutured wound edges; (f) making an incision in the shape of the m cocele using a CO2 laser; (g) removing a mucocele as in (c); (h) letting blood accumulate in the sha of the resection wound, and forming an artificial scab on the surface; (i) sagittal cross section of (h (j) performing PBMT; (k) high likelihood of healing without scar formation.

Figure 3 .
Figure 3. Removal of mucocele using a CO 2 laser: (a) before removal of mucocele; (b-e) conventional surgical method; (f-k) surgical method using CO 2 laser; (b) designing a spindle incision line over the mucocele along a lip wrinkle and making a submucosal incision; (c) removing a mucocele without damaging it; (d) hemostatic suturing; (e) high likelihood of formation of a linear scar and a mucosal concavity along the sutured wound edges; (f) making an incision in the shape of the mucocele using a CO 2 laser; (g) removing a mucocele as in (c); (h) letting blood accumulate in the shape of the resection wound, and forming an artificial scab on the surface; (i) sagittal cross section of (h); (j) performing PBMT; (k) high likelihood of healing without scar formation.

Figure 4 .
Figure 4. Treatment of open lip vermillion wounds using a CO2 laser: (a-c) procedures common to both hemostatic suturing and treatment with a CO2 laser; (d,e) treatment with hemostatic suturing only; (f-k) treatment using CO2 laser; (a) immediately after injury; (b) after debridement; (c) before hemostatic suturing; (d) suturing with prioritization of wound closure but without consideration of esthetic outcomes of the lip; (e) an interrupted inferior vermillion border of the upper lip, with a high possibility of healing with a linear scar; (f) minimum suturing to make an uninterrupted inferior vermillion border of the upper lip; (g) letting blood accumulate in the shape of a parenchymal defect in the tissue; (h) sagittal cross section of (g); (i) forming an artificial scab on the blood at the wound surface; (j) performing PBMT; (k) high likelihood of healing without mucosal scars.A dotted line indicates the inferior vermillion border of the lip.

Figure 4 .
Figure 4. Treatment of open lip vermillion wounds using a CO 2 laser: (a-c) procedures common to both hemostatic suturing and treatment with a CO 2 laser; (d,e) treatment with hemostatic suturing only; (f-k) treatment using CO 2 laser; (a) immediately after injury; (b) after debridement; (c) before hemostatic suturing; (d) suturing with prioritization of wound closure but without consideration of esthetic outcomes of the lip; (e) an interrupted inferior vermillion border of the upper lip, with a high possibility of healing with a linear scar; (f) minimum suturing to make an uninterrupted inferior vermillion border of the upper lip; (g) letting blood accumulate in the shape of a parenchymal defect in the tissue; (h) sagittal cross section of (g); (i) forming an artificial scab on the blood at the wound surface; (j) performing PBMT; (k) high likelihood of healing without mucosal scars.A dotted line indicates the inferior vermillion border of the lip.

Figure 5 .
Figure 5. Differences in the amount of blood accumulated in the tooth extraction socket due to different hemostasis methods: (a) suturing of the extraction wound; (b) compression hemostasis with a cotton roll; (c) blood coagulation by artificial scab using a CO2 laser.The broken line indicates the position of the blood surface layer within the tooth extraction socket.

Figure 5 .
Figure 5. Differences in the amount of blood accumulated in the tooth extraction socket due to different hemostasis methods: (a) suturing of the extraction wound; (b) compression hemostasis with a cotton roll; (c) blood coagulation by artificial scab using a CO 2 laser.The broken line indicates the position of the blood surface layer within the tooth extraction socket.

Figure 6 .
Figure 6.Comparison of tooth extraction wound healing between compression hemostasis and artificial scab formation with a CO2 laser: (a-e) the wound healing process after compression hemostasis with a cotton roll; (f-j) wound healing process with an artificial scab; (a,f) coagulation stage; (b,g) granulation tissue stage; (c,h) temporary bone stage; (d,i) healing stage; (e,j) histopathological image of a tooth extraction wound 7 days after tooth extraction in a rat (corresponds to the temporary bone stage in humans); (a) the amount of blood in the extraction socket is decreased due to blood absorption by dental cotton rolls; (b) the space-making effect of bone regeneration is decreased due to extension and invagination of mucosal epithelium surrounding the extraction wound into the extraction socket and depression of the extraction wound with contraction of the epithelial

Figure 6 .
Figure 6.Comparison of tooth extraction wound healing between compression hemostasis and artificial scab formation with a CO 2 laser: (a-e) the wound healing process after compression hemostasis with a cotton roll; (f-j) wound healing process with an artificial scab; (a,f) coagulation stage; (b,g) granulation tissue stage; (c,h) temporary bone stage; (d,i) healing stage; (e,j) histopathological image of a tooth extraction wound 7 days after tooth extraction in a rat (corresponds to the temporary bone stage in humans); (a) the amount of blood in the extraction socket is decreased due to blood absorption by dental cotton rolls; (b) the space-making effect of bone regeneration is decreased due to extension and

Figure 7 .
Figure 7. Free gingival graft using a CO2 laser: (a-e) donor site; (f-h) recipient site; (a) determine the area for a gingival flap; (b) harvest the gingival flap; (c) after harvesting the flap, let blood accumulate on the tissue surface of the donor site to the height of the surrounding mucosa; (d) form artificial scabs; (e) perform PBMT; (f) place the gingival flap firmly in the recipient site to avoid formation of dead space between them, and then suture the flap to the surrounding mucosa; (g) solder the wound edges in the spaces between suture points with HILT; (h) perform PBMT.

Figure 7 .
Figure 7. Free gingival graft using a CO 2 laser: (a-e) donor site; (f-h) recipient site; (a) determine the area for a gingival flap; (b) harvest the gingival flap; (c) after harvesting the flap, let blood accumulate on the tissue surface of the donor site to the height of the surrounding mucosa; (d) form artificial scabs; (e) perform PBMT; (f) place the gingival flap firmly in the recipient site to avoid formation of dead space between them, and then suture the flap to the surrounding mucosa; (g) solder the wound edges in the spaces between suture points with HILT; (h) perform PBMT.
[26,27]ation of mucosal epithelium surrounding the extraction wound into the extraction socket and depression of the extraction wound with contraction of the epithelial scar; (c) new bone is regenerated from the bottom of the extraction socket; (d) alveolar bone height is decreased and a dish-like concavity forms in the mucosa of the extraction wound; (e) histopathological image of a rat tooth extraction socket at the temporary bone stage after compression hemostasis; (f) formation of an artificial scab lets blood accumulate to the height of the mucosa around the extraction wound, followed by PBMT; (g) the space-making effect for bone regeneration by the artificial scab prevents extension and invagination of mucosal epithelium around the extraction wound into the extraction socket, in combination with suppression of contraction of the mucosal epithelial scar by PBMT.Also, due to the healing-promoting effect of PBMT, the appearance of cells involved in bone regeneration in the shallow layer of the extraction socket and the production of collagen fibers were promoted, followed by the migration of osteoblasts on those collagen fibers; (h) new bone forms with a cross-linking pattern in the shallow and middle layers of the extraction socket, in addition to bone regeneration from the bottom of the extraction socket; (i) alveolar crest height is preserved and there is no concavity of the mucosal epithelium, due to the newly generated bone with a cross-linking pattern that serves as a bone lining under the mucosal epithelium of the extraction wound; (j) histopathological image of a rat tooth extraction socket in temporary bone stage after covering with artificial scab.The broken line in (e) and (j) indicates the height of the alveolar bone.Reproduced from[26,27]under Creative Commons CC BY-NC 4.0.