PERIAPICAL WOUND HEALING

PERIAPICAL WOUND HEALING

1. PERIAPICAL WOUND HEALING

2. Wound healing is the response of living tissues to any injury which causes disruption of the continuity and/or function of those tissues and involves a complex series of biological events, some occurring simultaneously and others dependent upon the completion of prior events. Wound healing is basically dependent upon the type of tissue wounded and the type of wound that the tissue receives The tissues wounded in periradicular surgery are the mucoperiosteal tissues (gingiva, alveolar mucosa, palatal mucosa, and underlying periosteum), periradicular tissues (bone, gingival ligament, and periodontal ligament), and radicular tissues (cementum and dentin). These tissues, with the exception of dentin, are collectively termed the periodontium and form the supporting structures of the teeth. The tissues of the periodontium receive three types of surgical wounding during periradicular surgery: incisional wounding, blunt dissectional wounding, and excisional wounding. Incisional wounds are made with a scalpel, outline the perimeter of the flap, and involve the mucoperiosteal tissues. Blunt dissectional wounds are made with a periosteal elevator, separating mucoperiosteal tissues from cortical bone during the flap reflection procedure. Excisional wounds are made with a rotary instrument in removing bone and resecting the root end . Thus, with multiple types of oral tissues receiving various types of surgical wounding, the entire wound healing response to periradicular surgery is understandably diverse and complicated.

3. Connective tissue involves a process of repair via the formation of granulation tissue, whereas non-connective tissue, such as that of the glandular organs, smooth muscles, skeletal muscles and peripheral nerves, involves the proliferation, and therefore, regeneration, of the remaining tissue18. These two processes are dependent upon the regenerative capacity of the affected cells, the extent of the affected site, and the proliferative activity of the stromal tissue. Regeneration involves a process of tissue renewal with cells that have similar characteristics to those that were previously lost; it is the morphological and functional restoration of tissue. Conversely, repair is characterized by the formation of connective tissue at the site of the lesion, During pulp infection, the occluded blood supply of the root canal becomes conducive to bacterial periapical region is elicited to neutralize the antigen. immune cells, which are then organized into a barrier to sequester the infection. Bone resorption and bone formation are processes involving the activity of osteoclasts, osteoblasts, and osteocytes; they are affected by the systemic and local conditions66. However, bone homeostasis is disrupted during apical periodontitis, which promotes increased rates of bone resorption

4. Wound healing is the host’s programmed immunoinflammatory defense mechanism in response to infection or injury The primary difference is that healing after surgery requires a blood clot formation. Surgical excision may result in a faster healing process compared to NSRCT, which exhibits slower healing dynamics. After successful NSRCT, periapical inflammatory tissues will be eliminated, mainly by phagocytic debridement. The basic phases of wound healing can be divided into three overlapping stages: inflammation, proliferation, and remodeling. Within these three broad phases a complex and coordinated series of events occur that include chemotaxis and phagocytosis during the inflammatory phase. Neocollagenesis , epithelialization, and angiogenesis result in the formation of granulation tissue during the proliferation phase. During the final remodeling phase, there is active collagen remodeling and tissue maturation that culminates in either repair or regeneration

5. Healing after Apical Microsurgery (1) osseous healing involving trabecular and cortical bone and (2) dentoalveolar healing that results in repair or regeneration of apical attachment apparatus (alveolar bone, periodontal ligament, and cementum) After apical surgery, the resected cavity is occupied by a coagulum, which is slowly replaced by granulation tissue originating from the periodontal ligament and endosteum. The formation of new bone begins in the internal area and progresses externally toward the level of the former cortical plate. As newly laid woven bone reaches the lamina propria, the overlying membrane becomes functional periodontium (osseous healing) (Figure 13.3). Progenitor cells from the periodontal ligament differentiate into periodontal ligament cells and cementoblasts to cover the resected root surface and lead to regeneration of the cementum and the periodontal ligament (dentoalveolar healing

6. Incomplete Healing/Scar Formation Scar tissue formation after apical surgery has been extensively studied. It was demonstrated that 26% of defects radiographically larger than 10 mm resulted in scar formation after apical surgery. Furthermore, when the bony defect perforated both cortical plates (“through-and-through lesion”), the incidence of scar tissue formation may reach 60%. However, there is a lack of clinical evidence to indicate that large or through-and-through lesions will always result in scar tissue formation, even when no barrier membrane is used after apical surgery

7. Radiographically, scar formation has been characterized by a typical sunburst appearance due to bone trabeculae radiating from a center of the lesion that may remain radiolucent indefinitely (Figure 13.4). To this day the mechanisms of scar formation are not fully understood. Nevertheless, during incomplete healing, the healing is periosteal in nature and progresses from the outside of the lesion towards the inside, resulting in a residual defect and accumulation of nonfunctional fibrous tissue.

8. In this context, the study conducted by Kerekes and Tronstad (1979) should also be mentioned, as the authors reported an 85% success rate with complete healing and 6% still undergoing healing in the treatment of teeth with apical periodontitis, without using calcium hydroxide. The healing process is known to be quicker and the recovery complete in young people compared to older people. Similar studies to the present one indicated that age is an important factor in the healing of periapical lesions in patients aged between 11 and 24 years ( Gesi et al 2006). It is assumed that periapical lesions occur due to the immune response to antigens from the root canal. Their interaction with antibodies causes immunological reactions in periapical tissues. Immunological reactions involve the removal of invasive antigenic substances. The healing of periapical structures depends on their ability to develop an immune response to the action of various antigens. The presence of immunocompetent cells, especially T cells, in periapical lesions indicates the presence of humoral immune responses in that area. These complex immune responses play an important role in periapical lesions. However, immune functions are weakened when reaching sexual maturity due to changes in the number and proliferation of immunocompetent cells. The decrease in T-cell proliferation in response to antigens, together with the poor reaction of mature T cells to antigens, are the main factors associated with lower immune responses associated with increased age (Matsuo et al 1994, Kurashima & Utsuyama 1997).

9. In contrast, in a study that included patients aged between 19 and 86 years, Peters and Wesselink (2002) reported that there is no connection between patient age and periapical healing. In their study, they obtained a 71% success rate of root canal therapy performed in two sessions, after a period of 4½ years. These observations are consistent with studies showing no significant difference in periapical tissue response when T cell functions have been suppressed (Peters & Wesselink 2002), similar to elderly patients. According to this conclusion, we can say that immunodeficiency is not a significant factor in the healing of periapical lesions in the elderly. Due to the controversy over the influence of age on periodontal healing, there is a need for more comparative studies. Khabbaz and Papadopoulos (1999) have determined that periapical wound healing is not affected by the presence of root canal filling material in the periapical tissue. They also concluded that healing is due to infection control during root canal preparation and, equally, during root canal filling. Katebzadeh et al (2000) also mention the importance of root canal filling in the healing of apical periodontitis. Failure due to overfilling is actually caused by infected dentin and debris pushed beyond the apex during instrumentation. Augsberger and Peters (1990) stressed that periapical would healing takes place even if the root canal sealer reaches the periapical tissue, and possible failure is due to other factors, such as improper handling. Lin et al. (1992) stressed that root canal filling material has a much lower irritant effect than microbial factors. Huang et al (2002) have concluded that the biocompatibility of the root canal sealer is extremely important as it stimulates the reorganization of the affected periapical tissue that it comes into contact with. Tanomaru et al (1998) stated that in case of teeth with chronic periapical infection, root canal filling materials with antibacterial properties that do not irritate the periapical or periradicular tissues can stimulate apexification

10. Leonardo et al (2003) showed that periapical tissue reaction is excellent in the presence of AH Plus. In their study, they observed the presence of mineralized tissue apposition in the apical zone of the root and, in many cases, soft tissue mineralization processes around the apex. Azar et al (2000) have found that freshly mixed AH Plus has mutagenic and cytotoxic properties, which disappear when filling is completed. Dartar et al (2003) reported the lack of AH Plus cytotoxicity in vitro and the promotion of bone healing. Other studies have shown that the type and amount of root canal filling material used, its resorption capability and especially its toxicity are all important factors in the healing of periapical lesions. Another interesting aspect of periapical healing is the link between the healing process and the position of the tooth in the dental arch.

11. It seems that maxillary second premolars as well as maxillary and mandibular canines have a better prognosis than other teeth. The first maxillary molars often have two middle-vestibular root canals, and one of them could remain unspotted, untreated and unfilled, thus maintaining the infection active. Maxillary lateral incisors present anatomical variations, such as pronounced curvature in the apical area and very thin roots, aggravating the correct mechanicaland antiseptic treatment as well as the root canal filling process. However, other studies suggest that there is no connection between periapical healing and tooth position in the dental arch ( Orstavik & Horsted-Bindslev 1993)

12. Healing Evaluation Using CBCT it illustrates the defects in cancellous bone and cortical bone separately, making it a more sensitive tool to identify apical periodontitis. Furthermore, image reconstruction occurs in a multiplanar reformation mode, which allows the highlighting of specific anatomic regions and structures around the resected root surface, such as periodontal ligament space, lamina dura, and cortical plate. Moreover, it allows for differentiation between various bone densities. The aforementioned anatomic structures are central to current criteria used to assess apical surgery healing on CBCT. For example, complete healing is attributed to those cases where the periodontal ligament space and the lamina dura have completely reformed over the resected root surfaces Other interesting healing patterns observed on CBCT include complete healing in the immediate vicinity of the resected root surface, along with a complete cortical bone repair in width and density; however, the trabecular bone adjacent to the resected root is of low density These cases can be attributed to limited healing, which is considered a successful outcome. This differentiation between various bone densities is unique to CBCT. It has been hypothesized that reduced radiolucent areas represent either scar tissue, immature bone, or bone-like tissue without adequate mineralization, which at this particular stage of healing would not be radiopaque enough to be detected by CBCT. Another interesting observation concerns tooth position in the bony architecture. This is where presurgical assessment using CBCT becomes an essential step in treatment planning. It was noted that there was superior healing in teeth positioned deeper in the dental architecture (i.e., surrounded with bone except in the apical area, where there was radiolucency and fenestration of the cortical plate) than in teeth positioned far buccally, where roots were too prominent and a very thin cortical plate covered them, even though there was no fenestration of the cortical plate (Figure 13.7). In these cases, the placement of a bone graft and/or a collagen-based membrane can help in the healing process. The bone graft will permit thickening of the cortical plate, whereas the collagen membrane will contain the bone graft material and exclude epithelial cells from penetrating the osteotomy site. Recent developments in grafting material suggest the usage of a collagen-based augmentation material that functions as both a bone graft and membrane

13. Ingrida Dapkute et al Periapical Wound Healing Microsurgery in Endodontics, First Edition. Syngcuk Kim and Samuel Kratchman . © 2018 John Wiley & Sons, Inc. Published 2018 by John Wiley & Sons, Inc.

14. The healing of periapical structures depends on their ability to develop an immune response to the action of various antigens. The presence of immunocompetent cells, especially T cells, in periapical lesions indicates the presence of humoral immune responses in that area. These complex immune responses play an important role in periapical lesions. However, immune functions are weakened when reaching sexual maturity due to changes in the number and proliferation of immunocompetent cells. The decrease in T-cell proliferation in response to antigens, together with the poor reaction of mature T cells to antigens, are the main factors associated with lower immune responses associated with increased age (Matsuo et al 1994, Kurashima & Utsuyama 1997). Due to the controversy over the influence of age on periodontal healing, there is a need for more comparative studies. Khabbaz and Papadopoulos (1999) have determined that periapical wound healing is not affected by the presence of root canal filling material in the periapical tissue. They also concluded that healing is due to infection control during root canal preparation and, equally, during root canal filling. Katebzadeh et al (2000) also mention the importance of root canal filling in the healing of apical periodontitis. Failure due to overfilling is actually caused by infected dentin and debris pushed beyond the apex during instrumentation.

15. Augsberger and Peters (1990) stressed that periapical would healing takes place even if the root canal sealer reaches the periapical tissue, and possible failure is due to other factors, such as improper handling. Lin et al. (1992) stressed that root canal filling material has a much lower irritant effect than microbial factors. Huang et al (2002) have concluded that the biocompatibility of the root canal sealer is extremely important as it stimulates the reorganization of the affected periapical tissue that it comes into contact with. Tanomaru et al (1998) stated that in case of teeth with chronic periapical infection, root canal filling materials with antibacterial properties that do not irritate the periapical or periradicular tissues can stimulate apexification

16. and healing. Leonardo et al (2003) showed that periapical tissue reaction is excellent in the presence of AH Plus. In their study, they observed the presence of mineralized tissue apposition in the apical zone of the root and, in many cases, soft tissue mineralization processes around the apex. Azar et al (2000) have found that freshly mixed AH Plus has mutagenic and cytotoxic properties, which disappear when filling is completed. Dartar et al (2003) reported the lack of AH Plus cytotoxicity in vitro and the promotion of bone healing. Other studies have shown that the type and amount of root canal filling material used, its resorption capability and especially its toxicity are all important factors in the healing of periapical lesions. Another interesting aspect of periapical healing is the link between the healing process and the position of the tooth in the dental arch. It seems that maxillary second premolars as well as maxillary and mandibular canines have a better prognosis than other teeth. The first maxillary molars often have two middle-vestibular root canals, and one of them could remain unspotted, untreated and unfilled, thus maintaining the infection active. Maxillary lateral incisors present anatomical variations, such as pronounced curvature in the apical area and very thin roots, aggravating the correct mechanicaland antiseptic treatment as well as the root canal filling process. However, other studies suggest that there is no connection between periapical healing and tooth position in the dental arch ( Orstavik & Horsted-Bindslev 1993)

17. At the end of 12-month period, only 20 patients presented themselves for check-up. Therefore, only 22 teeth could be assessed, 11 treated with AH Plus (group A) and 11 with RealSeal SE (group B). Data regarding clinical signs and symptoms related were collected and recorded, compared with baseline values, but they were not subjected to statistical analysis.

18. PRINCIPLE OF PERIAPICAL WOUND HEALING It is a host’s ‘‘programmed event,’’ which begins with (1) hemostasis or coagulation phase (2) inflammation phase (3) proliferative phase (4) regeneration and/or repair phase (5) remodeling or maturation phase Regardless of the size of a wound, granulation tissue in the proliferative phase, a necessary element of wound healing, fills the wound and helps complete the wound healing process

19. Wound healing usually involves recruitment and differentiation of progenitor/stem cells into tissue committed cells . Wound healing can result in either regeneration or repair, depending on the nature of wound, availability of progenitor/stem cells, growth/differentiation factors, and microenvironmental cues such as adhesion molecules, extracellular matrix (ECM), and associated noncollagenous protein molecules Regeneration represents the replacement of damaged tissue by the cells of the same tissue. Importantly, it reconstitutes, although not completely, both the architecture and functions of the original tissue, such as healing of an uninfected simple surgical incision of the skin approximated by surgical sutures, because tissue destruction and granulation tissue formation are minimum

20. Regeneration of periapical tissues after periapical surgery requires recruitment of progenitor/stem cells to differentiate into committed osteoblasts, PDL cells, and cementoblasts growth/differentiation factors as necessary signals for attachment, migration, proliferation, and differentiation of progenitor/stem cells; local microenvironmental cues such as adhesion molecules, and ECM and associated noncollagenous protein molecules

21. Cementoblasts , PDL cells, and osteoblasts in the periapical tissues are differentiated cells, they still retain the potential to undergo cell division and proliferation on stimulation by appropriate signals during physiologic turnover and periapical wound healing In small periapical lesions, resident osteoblasts, PDL cells, and cementoblasts might be capable of restoring damaged periapical tissues. However, in large periapical lesions, periapical wound healing requires recruitment and differentiation of progenitor cells/stem cells into osteoblasts, cementoblasts , and PDL cells. It has been also shown that PDL harbors adult stem cells in the paravascular spaces, and these stem cells are capable of differentiating into PDL-like, cementoblastlike , and osteoblast-like cells . In addition, bone marrow mesenchymal stem cells and periosteal osteoprogenitor cells are capable of differentiating into osteoblasts

22. Cell differentiation is regulated by extrinsic local microenvironmental cues and intrinsic master regulatory genes . Cell differentiation is usually a part of the regenerative process . Regardless of the size of periapical lesions, persistence of root canal infection is the primary cause of inflamed periapical tissues not to heal after endodontic therapy . There are no published studies demonstrating that membrane barriers and/or bone grafts contributed to the cause of periapical surgery failure. Complete periapical wound healing after periapical surgery should include regeneration of alveolar bone, PDL, and cementum

23. In 1974, platelets regenerative potentiality was introduced, and Ross et al.,[3] were first to describe a growth factor from platelets. After activation of the platelets which are trapped within fibrin matrix, growth factors released and stimulate the mitogenic response in the bone periosteum during normal wound healing for repair of the bone.[4] Better understanding of physiologic properties of platelets in wound healing since last two decades led to increase its therapeutic applications in the various forms showing varying results.

24. Kim et al. 2012 May Animal study The PRF-mixed tricalcium phosphate (TCP) showed more rapid bone healing than the (recombinant human bone morphogenic protein 2) rhBMP-2-coated TCP or the TCP-only control Jankovic et al. 2012 Apr Randomized controlled clinical study Use of a PRF membrane in gingival recession treatment provided acceptable clinical results, followed by enhanced wound healing and decreased subjective patient discomfort compared to connective tissue graft (CTG)-treated gingival recessions Rudagi et al. 2012 Apr Case report This case report presents the successful healing and apexification with combined use of MTA as an apical barrier and autologous PRF membrane as an internal matrix

25. Pradeep et al. 2012 Mar Randomized control clinical trial Porous hydroxyapatite (HA) when added to PRF increases the regenerative effects observed with PRF in treatment of human three wall intrabony defects Anitua et al. 2012 Mar In vivo Practically, plasma rich in growth factors (PRGF) may present a role in reducing tissue inflammation after surgery, increasing new bone formation, and promoting vascularization of bone tissue Peck et al. 2012 Mar Case report L-PRF is a newly developed platelet concentrate that has successfully been used in a number of surgical procedures to optimize wound healing and was used to stimulate bone formation to facilitate ideal placement of implants Clipet et al. 2012 Feb In vitro PRF conditioned medium induced gene expression in osteoblasts. Expression of osteopontin and osteocalcin and late osteogenic markers was observed and confirmed PRF is useful in stimulating tissue healing and bone regeneration Jayalakshmi et al. 2012 Case report Combined use of PRF and b-tricalcium phosphate (b-TCP) for bone augmentation in treatment of periapical defects is a potential treatment alternative for faster healing than using these biomaterials alone

26. Simonpieri et al. 2009 Jun In vivo PRF membranes are particularly helpful for periosteum healing and maturation. The thick peri-implant gingival is as a result of several healing phases on a PRF membrane layer Simon et al. 2009 May Animal study PRFM alone may be the best graft for ridge preservation procedures Simonpieri et al. [13] 2009 Apr In vivo PRF membranes protects the surgical site, promotes soft tissue healing, and when its fragments mixed with graft material it functions as a “biological connector” Magremanne et al. 2009 Apr Case report PRF may induce healing of non- reossified cystic cavity by supplying local growth factors Aroca et al. [19] 2009 Feb In vivo Modified coronally advanced flap (MCAF) is a predictable treatment for multiple adjacent Miller Class I or II recession-type defects. The addition of a PRF membrane positioned under the MCAF provided inferior root coverage, but an additional gain in gingival/mucosal thickness (GTH) at 6 months compared to conventional therapy Anilkumar et al. [17] 2009 Jan Case report Described laterally displaced flap technique with PRF membrane technique as a navel root coverage approach for gingival recession of the mandibular anterior teeth Diss et al. [85] 2008 May In vivo The bone-added osteotome sinus floor elevation (BAOSFE) procedure with PRF as grafting material can lead to an endosinus bone gain Lundquist et al. [86] 2008 May Review PRF provides sustained release and protection against proteolytic degradation of endogenous fibrogenic factors important for wound healing

27. PLATELET RICH PLASMA PRP blood clot, on the other hand, contains 4% RBCs, 95% platelets, and 1% WBCs. The PRP preparation protocol requires collection of blood with anticoagulant, centrifugation in two steps, and induced polymerization of the platelet concentrate using calcium chloride and bovine thrombin. PRP has been used in conjunction with different grafting materials in bone augmentation procedures since the day of its introduction

28. PRF Choukroun developed the PRF in 2001 at France Actually the platelets and leukocyte cytokines are important part in role play of this biomaterial, but the fibrin matrix supporting them is very helpful in constituting the determining elements responsible for real therapeutic potential of PRF. Cytokines are immediately used and destroyed in a healing wound. The harmony between cytokines and their supporting fibrin matrix has much more unique importance than any other constant. A fibrin glue, enriched with cytokines (such as PRP) with large uncontrollable and short-term effect is less better than a physiologic fibrin matrix (such as PRF) with good and better effects. Naik, et al.: Healing potential of PRF Journal of Conservative Dentistry | Jul-Aug 2013 | Vol 16 | Issue 4 289 to accumulate platelets and released cytokines in a fibrin clot.

29. Choukroun attempted to evaluate the potential of PRF in combination with freeze-dried bone allograft (FDBA) in sinus floor elevation to enhance bone regeneration ,nine sinus floor augmentations were performed. Out of nine; in six sites, FDBA with PRF (test group), and in three sites FDBA without PRF (control group) was used. After 4 months, the test group and for the control group after 8 months; bone specimens from the augmented region during the implant insertion procedure were harvested and evaluated. After 4 months of healing time, histologic maturation of the test group appears to be identical to that of the control group which was for a period of 8 months with equivalent quantities for both protocols. In various bone reconstruction procedures Choukroun’s PRF could provide a possible new bone. Mazor stated that use of PRF as the sole filling material during a simultaneous sinus lift and implantation procedure had stabilized a good amount of regenerated bone in the subsinus cavity up to the tip of implants in a case series through a radiological and histological evaluation at after 6 months from the surgery. Also they advocated that Choukroun’s PRF, which is a simple and inexpensive biomaterial in systematic use during a sinus lift seems as an acceptable option.

30. PRF membranes protects the surgical site; promotes soft tissue healing; and when its fragments mixes with graft material, it functions as a “biological connector” between the different elements of graft and acts as a matrix which supports neoangiogenesis , capture of stem cells, and migration of osteoprogenitor cells to the center of graft. PRF plugs can also be used in treating the residual extraction sockets. Use of autologous PRF in extracted socket filling after immediate bone augmentation using titanium membranes applied to the socket walls and primary closure was found to be feasible and safe with adequate bone filling after 8 weeks or above for implant fixation. Anilkumar reported PRF as a potential novel root coverage approach for treating gingival recession in mandibular anterior teeth using combined laterally positioned flap technique and PRF membrane. Combined use of PRF and bone graft with good results has also been reported for combined periodontic-endodontic furcation defect.

31. PRF as a scaffolding material in an infected necrotic immature tooth for pulpal regeneration and tooth revitalization as it satisfies many criteria of an ideal physical scaffold. Another advantage of using PRF as a scaffold is that it has a trimolecular or equilateral fibrin branch junction which makes its architecture flexible and can support cytokine enmeshment and cellular migration. 20 ml of 5.25% sodium hypochlorite was used to irrigate the canal. Care was taken to ensure that the irrigating needle was loose in the canal and that the NaOCl irrigation was performed very slowly. Triple antibiotic paste was used for disinfection of the canal because this particular combination is effective in addressing the diverse flora present in the root canal. Sato et al, investigated this drug combination in vitro and found it to be very effective in the sterilization of carious lesions, necrotic pulps, infected root dentin and periapical lesions. This drug combination is also effective in killing the bacteria in the deep layers of root canal dentin

32. Directly over the PRF clot the MTA was packed and condensed to obtain a tight coronal seal as it is hydrophilic and needs moisture to set, which is a favorable property when there is potential for moisture contamination in the clinical setting, and also MTA by itself provides signaling molecules for the growth of the stem cells. The positive response to cold test and EPT testing in our case report can be attributed to the placement of MTA slightly below the level of CEJ. If we had got negative response to the vitality testing it could have been due to the thickness of MTA which halts the growth of the new tissue ahead of it and also if placement of MTA is near CEJ elicits a higher positive response.

33. Bone grafting materials include autografts, allografts, xenografts, and alloplasts . They have been used in periodontal regenerative therapy as space maintainers for selective denuded root surfaces or to act as osteoinductive or osteoconductive biomaterials for regeneration of bone loss as a result of periodontal disease . Bone grafts have also been successfully used to regenerate new bone formation in implant dentistry . The same bone grafting materials, especially alloplasts such as calcium sulfate, have been widely used in periapical surgery to enhance new bone formation as well. Calcium sulfate must dissolve in tissue fluid or integrate into bone before or during new bone formation. Similar to periodontal regenerative therapy, evaluation of wound healing after periapical surgery by using bone grafts should also include regeneration of PDL

34. Most bone grafts, especially calcium sulfate used in periapical surgery, are neither osteogenic nor osteoinductive Therefore, calcium sulfate is not capable of recruiting mesenchymal stem cells in the bone marrow or endosteum and osteoprogenitor cells in the periosteum to differentiate into committed pre-osteoblasts. Calcium sulfate is osteoconductive which refers to the ability of some foreign materials to serve as a scaffold on which cells can attach, migrate, and grow and divide . Even though bone grafts are osteoconductive, they are not ideal materials for promoting periodontal tissue regeneration such as PDL and cementum in periodontal regenerative therapy because they are not able to stimulate the formation of a new connective tissue attachment. Many studies have clearly demonstrated that calcium sulfate can serve as scaffold for new bone formation in periapical surgery.

35. Biologically, a blood clot is a better space filler or ECM than all bone grafting materials. A blood clot is the host’s own biologic product and is essential to tissue wound healing. Without a blood clot, tissue wound healing would be impaired as in a dry socket after tooth extraction. A blood clot is composed of insoluble fibrin and many growth factors/cytokines such as platelet-derived growth factor (PDGF), TGF-b, vascular endothelial growth factor (VEGF), endothelial growth factor, insulin-like growth factor (IGF), and fibroblast growth factor (FGF) . During wound healing, fibrin filaments cross-linked to fibronectin provide a provisional matrix for attachment and migration of immune cells, fibroblasts, endothelial cells, and tissue cells . The degraded products of fibrin, by plasmin, are chemotactic to the host’s immune cells . In addition, FGF, TGF-b, VEGF, and endothelial growth factor in blood clot promote angiogenesis to enhance tissue wound healing . Bone grafts alone without a blood clot or angiogenic factors are unlikely to be capable of promoting periapical wound healing

36. CON PRF can be used to promote wound healing, bone regeneration, graft stabilization, wound sealing, and hemostasis. Because the fibrin matrix is better organized, it is able to more efficiently direct stem cell migration Release of growth factors from PRF through in vitro studies and good results from in vivo studies led to optimize the clinical application of PRF. It was shown that there are better results of PRF over PRP. Dohan proved a slower release of growth factors from PRF than PRP and observed better healing properties with PRF. It was observed and shown that the cells are able to migrate from fibrin scaffold; while some authors demonstrated the PRF as a supportive matrix for bone morphogenetic protein a

37. Growth factors/cytokines play a crucial role in tissue wound healing because they regulate immune function and proliferation and differentiation of cells participating in wound healing . Growth factors are multifunctional and often have more than 1 target cell . Many of the host’s natural growth factors have been synthesized in vitro and used alone or incorporated into bone grafts in periapical surgery to enhance new bone formation. In a clinical study, combination of platelet-rich plasma and tricalcium phosphate placed in a bony defect after periapical surgery was shown to enhance bone regeneration . However, when exogenous recombinant human bone morphogenetic protein-1 (rhOP-1) (95), rhBMP-2 (96), IGF combined with PDGF, or FGF alone was delivered to the bony defect during periapical surgery, the growth/differentiation factors did not demonstrate any obvious benefit to the process of bone healing. The concentration and stability of exogenous growth factor/factors and their presence in relation to the temporal and spatial expression of other growth/differentiation factors as well as their exact target cells are important in tissue wound healing

38. Factors Influencing Periapical Wound Healing Numerous factors such as infection, foreign bodies, systemic disease, and an impaired host’s immune system can influence wound healing.Infection and foreign bodies are the most important factors that can affect periapical wound healing. Implanted biomaterials such as bone grafts, despite being inert and nontoxic, often trigger adverse foreign body reactions such as inflammation, fibrosis, infection, and thrombosis . The foreign body reaction composed of activated macrophages and foreign body giant cells is the end-stage response of inflammation and wound healing after implantation of biomaterials. Foreign bodies favor infection due to biofilm formation . In addition, any foreign materials such as bone grafts have to dissolve in tissue fluid or be phagocytosed by activated macrophages before wound healing can be completed. If that does not occur, bone grafts will be surrounded by fibrous connective tissue or embedded in newly formed bone, as in some instances of periodontal regenerative therapy

39. 9-year-old boy came with the chief complaint of broken upper front tooth (#8) along with discoloration. Past dental history revealed trauma to his upper front tooth (#8). The medical history of the patient was noncontributory. Intraoral examination of his teeth revealed the presence of discolored tooth #8 along with Ellis class IV fracture. Tooth #8 was sensitive to both percussion and periapical palpation tests. It did not respond to CO2 ice and electric pulp test (EPT). Periodontal probing depth of the tooth #8 was within normal limit. Intraoral Periapical Radiographic examination of tooth #8 revealed an immature root and an open apex associated with periapical radiolucency . Further radiographic examination of the tooth revealed a 3 mm open apex along with thin dentinal walls that appeared prone to fracture. So a clinical decision of performing a regenerative endodontic treatment using Choukroun’s Platelet Rich Fibrin was decided.

40. A written informed consent was obtained from the patient’s mother. Local anesthesia was achieved using Lignocaine (1:100000 adrenaline. After the rubber dam application, access cavity preparation was done on the tooth #8. The canal was thoroughly irrigated with 20 ml of 5.25% sodium hypochlorite solution (Novo Dental Product, India) and nuetralised with saline. Following this, irrigation was done using 10ml of hexidine solution (0.2% Chlorhexidine, Vishal Dentocare , India) and dried with paper points (Dentsply Maillefer Ballaigues ).

41. A mixture of Ciprofloxacin ( Cifran 500mg, Ranbaxy Lab, India), Metronidazole ( Metrogyl 400mg, J.B.Chemicals and Pharmaceuticals, India), and Minocycline paste ( Minoz 50 mg, Ranbaxy Lab, India) was prepared into a creamy consistency and introduced into the canal using a lentulospiral . A cotton pellet was placed and the cavity was temporarily sealed with cavit (Dental Products of India, India). The patient returned after 21 days to the clinic and was asymptomatic. Local anesthesia was given, followed by rubber dam isolation; then the access cavity was reopened and thoroughly irrigated with sterile saline solution and dried with paper points. A 12ml sample of whole blood was drawn intravenously from the patient’s right antecubital vein and centrifuged (REMI Model R-8c with 12×15ml swing out head) under 3000 rpm for 10 minutes to obtain the PRF which was jelly like in consistency. The PRF was condensed into the canal using a finger plugger (Dentsply Maillefer Ballaigues ) till the level the cementoenamel junction. Grey MTA ( ProRoot MTA; Dentsply) was placed directly over the PRF to a thickness of 3mm followed by a wet cotton pellet and cavit .

42. The patient was recalled after 3 days and the setting of MTA was confirmed. The access cavity was then double sealed with GIC and Composite restoration . The patient returned to the clinic after 3 months, 6 months, 9 months and 1 year for review and was asymptomatic; the tooth #8 showed negative response to percussion and palpation tests and responded positive to CO2 ice or an electric pulp tester (EPT). Radiograph revealed continued thickening of the dentinal walls, root lengthening, regression of the periapical lesion and apical closure

43. Based on the clinical and radiographic examination we can only say with certainty that the pulp space had returned to a vital state. Based on research in avulsed teeth and on a recent study on infected teeth, it is more likely that the tissue in the pulp space is more similar to periodontal ligament than to pulp tissue. The potential theory behind the success of the presented case could be attributed to a study conducted by Huang et al, who concluded that the PRF causes proliferation of human Dental Pulp Cells and increases the protein expression of osteoprotegerin (OPG) and alkaline phosphatase (ALP) activity. Some amounts of human dental pulp cells present in the apical papilla usually remain vital even in case of a large periapical lesion. After the regression of the inflammation and under the influence of Hertwigs Epithelial Root Sheath these Dental Pulp Cells differentiate into odontoblasts like cells. OPG and ALP expression are generally regarded as markers of odontoblastic differentiation. As there was no bleeding in the root canal before placing the PRF we conclude that whatever tissue was produced in the canal could be attributed to the presence of PRF. On the basis of the results obtained in our case report we conclude that revitalization of necrotic infected immature tooth is possible under conditions of total canal disinfection and PRF is an ideal biomaterial for pulp-dentin complex regeneration

44. Periapical tssue reactions, such as inflammatory cell infiltration, bone resorption and epithelial proliferation in apical periodontitis are the products of root canal infection. After surgical endodontic therapy, periapical wound healing should follow exactly the same course as that of nonsurgical endodontic therapy. The only difference is that surgical endodontic therapy will heal faster than nonsurgical endodontic therapy because of more effective artificial debridement of infected or wounded periapical tissues by surgical procedures as compared with biological debridement by phagocytes in nonsurgical procedures . Grupe et al. suggested that the epithelial cells of apical cysts are capable of division and proliferation by virtue of their ability to undertake anaerobic glycolysis. There is no evidence that epithelial cells in inflammatory apical cysts behave like malignant neoplastic cells, which are encoded with oncogenes and can self divide in the absence of appropriate extracellular signals, such as mitogens, cyclins, or cyclin-dependent protein kinases The remnants of epithelium left in the periapical tissues after surgical endodontic procedures will regress by programmed cell death similar to that of nonsurgical endodontic therapy if irritants have been removed

45. METHODS FOR NONSURGICAL MANAGMENT OF PERIAPICAL LESIONS Conservative root canal treatment without adjunctive therapy Bhaskar has suggested that instrumentation should be carried 1 mm beyond the apical foramen when a periapical lesion is evident on a radiograph. This may cause transitory inflammation and ulceration of the epithelial lining resulting in resolution of the cyst Bender in his commentary on Bhaskar’s hypothesis has added that penetration of the apical area to the center of the radiolucency establishes drainage and relieves pressure. Once the drainage stops, fibroblasts begin to proliferate and deposit collagen; this compresses the capillary network, and the epithelial cells are thus starved, undergo degeneration, and are engulfed by the macrophages. Although this proves to be an effective method Shah suggests the possibility that quiescent epithelial cells may be stimulated by instrumentation in the apical region, with resultant proliferation and cyst formation, and thus stressed on the need for follow-up for a period of at least two years.[

46. Healing of large cysts like well-defined radiolucencies following conservative root canal treatment has been reported. Although the cystic fluid contains cholesterol crystals, weekly debridement and drying of the canals over a period of two to three weeks, followed by obturation has led to a complete resolution of lesions by 12 to 15 months. Decompression technique The decompression technique involves placement of a drain into the lesion, regular irrigation, periodic length adjustment, and maintenance of the drain, for various periods of time. The drain could either be ‘I’ shaped pieces of rubber dam,polyethylene tube along with a stent, hollow tubes, a polyvinyl tubing,uction catheter[32] or a radiopaque latex tubing.There is no standard protocol as to the length of time necessary to leave the drain. It may be different for different kinds, sizes or locations of lesions.It can vary between two days to five years. Daily irrigation of the lesion can be carried out by the patient through the lumen of the drain using 0.12% chlorhexidine. The advantages of this technique are; it is a simple procedure, it minimizes the risk of damaging adjacent vital structures, and is easily tolerated by the patient. However, several disadvantages have also been noted; patient compliance is very essential, inflammation of the alveolar mucosa, persistence of the surgical defect at site, development of an acute or chronic infection, displacement or submergence of the drainage tube Rees suggests placement of a small amount of red wax over the end of the drain to prevent ulceration of the labial or buccal mucosa adjacent to the drain. The decompression technique is contraindicated in cases of large dental granulomas or any solid cellular lesion, assince there is an absence of a fluid-filled cavity to decompress.Active nonsurgical decompression technique This technique uses the Endo- eze vacuum system ( Ultradent , Salt Lake, Utah) to create a negative pressure, which results in the decompression of large periapical lesions.

47. NANOCRYSTALLINE HYDROXYAPATITE GRAFT nanocrystalline apatites are nonstoichiometric, calcium- (and OH-) deficient. They may incorporate substituted ions in their nano-sized particles . Specifically, their higher solubility accounts for calcium and hydroxide deficiencies than hydroxyapatite. Moreover, they are capable of being mature when exposed to humid environment.. A crucial biological function in bone depends on the small size and non-stoichiometry of apatite nanocrystals. These nanocrystals probably cause mineral phase with the solubility needed for resorption of the bone by osteoclasts. Therefore, they enable bone mineral to act as an ion ‘reservoir’ capable of either capturing or releasing ions (or small molecules) under the control of the body to ensure homeostasis. Given these unique features, bone is a living tissue, not an inert, which continuously undergoes remodelling and repairing processes. Synthetic apatite demonstrates good biological properties including biocompatibility, bioactivity, lack of toxicity or inflammatory, immunity reactions, and a relatively high bioresorbability .

48. Different synthetic ways have been utilized to prepare nano-sized apatite crystals. Yet, preparation of actual biomimetic nanocrystalline apatites might be considered as a scientific and technological challenge. Thus, the large surface- tovolume ratio, the existence of a surface hydrated layer, and non- apatitic in nature are important in the formation procedure of a solution. This layer is bound to disappear progressively as the stable apatite domains (in the core of the crystals) improve with time. Possessing a great ionic mobility, ion exchange and adsorption capacities allows for participation of this hydrated layer in the interaction with macromolecules . Effects of n-HA on epithelial cells Kawai and colleagues stated that n-HA might have a therapeutic effect on periodontal epithelium. Therefore, they conjectured that healing process of open wound by contraction effect could be increased through intravenous calcium-based nanoparticles. Role of n-HA in differentiation and proliferation of periodontal ligament (PDL) cells Kanaya and co-workers [14] observed n-HA could stimulate differentiation of PDL cells, mediated by mechanosensitive signalling pathway and expression of BMP-2. Besides, Yang et al. [15] conducted an animal study through which they found n-HA could be used as a coating on silk scaffolds. Thus, they pointed out that n- HAcoated silk scaffolds might be potentially good biomaterials for regenerating periodontal tissue. Along with the above-mentioned studies, there are several research articles in the extant literature that emphasize n-HA effects on different cells in the periodontium.

49. Fibroblast Based on the results of a study by Saleh et al. , it was proven that silver n-HA could enhance fibroblast cell maturation and proliferation. This could eventually result in connective tissue regeneration. In contrast, n-HA was found to be much more biocompatible than silver nanomaterial in a study of evaluating the biocompatibility of silver and n-HA on fibroblast cells by Shahoon et al.. An in vitro study by Sun and colleagues revealed that n-HA could increase proliferation and differentiation of PDL fibroblast cells in comparison to dense hydroxyapatite. Additionally, it was pointed out that n-HA was more biocompatible than dense HA. Osteoblast Shnettler et al. found that n-HA could bind to the bone and stimulate the osteoblasts in the early stage of periodontal defect repair. This can lead to bone formation. Similar results were found in a study by Thian and co-workers . Moreover, Pilloni et al. proved that n-HA can increase the proliferation and differentiation of osteoblasts. In a report by Webster et al. greater protein adsorption and osteoblastic cells adhesion on n-HA were shown. Liu et al. [23] found that n-HA could stimulate binding and proliferation of osteoblast-like MG-63 cells. It was proven that n-HA exhibits biocompatibility and minimal toxic effect on osteoblast cells in studies by Motskin et al. Hsieh et al. , and Zhao et al. . Osteoclast In a study by Detsch et al. it was shown that n-HA with low or no carbonate content can enhance the differentiation of osteoclast-like cells. This can result in having a great number of osteoclast cells on the material compared to carbonate-rich group. Activated osteoclast recruited mesenchymal cells from the bone marrow to differentiate them into osteoblasts.

50. Effects of n-HA on bone regeneration Jahangirnezhad et al. reported that n-HA contains osteoconductive properties which make it capable of producing sufficient amount of bone as bone grafting material. Similarly Vullo et al. indicated that n-HA possesses both osteoconductive and osteoinductive properties in periodontal defects in dogs. Gotz et al. evaluated the immunohistochemical properties of hydroxyapatite nanocrystalline silica gel on biopsies obtained from jaw bone. The results revealed n-HA had osteoconductive and biomimetic properties. These properties were integrated into human physiological bone turnover at an early stage. By obtaining clinical results which were comparable to autogenous graft materials Huber et al. concluded that n-HA paste was appropriate for filling bone defects. Based on a study by Talal et al. n-HA-polylactic acid composite may be a suitable graft material for guided tissue regeneration (GTR) membrane. Although this material acts as a barrier, it can enhance bone regeneration via delivery of biologically active molecules. These results were supported in a study by Busen et al. [where they found n-HA could compete with Bio-Oss in bone reconstruction surgeries . They found that bone formation in Bio-Oss group was greater than n-HA

51. A 33-years-old female patient reported to the Department of Conservative Dentistry and Endodontics with a chief complaint of swelling on the inner surface of gum region in relation to upper front teeth for the past 10 days. Swelling was initially small, then gradually progressed and was associated with discomfort while taking food. Clinical examination revealed Ellis Class IV fracture in 21 with a swelling of 2.5 cm × 2 cm size seen over the palatal mucosa in relation to 21, 22, and 23 [Figure 1a]. Tenderness was felt on palpation over the apical mucosa in relation to 21, 22, and 23. These three teeth were sensitive to percussion tests. Pulp sensitivity tests revealed that 11, 21, 22, and 23 were nonvital. Preoperative intraoral periapical radiograph of 21, 22, and 23 reveals presence of large irregular periapical radiolucency (3 cm × 2 cm in size) at the apex of 21, 22, and 23 [Figure 2a and b]. This case was planned for conventional root canal treatment followed by periapical surgery. The root canal treatment was performed using step back technique till an apical size of #50, # 55, #45, and #60 in relation to teeth 11, 21, 22, and 23, respectively. .

52. Sodium hypochlorite (5.25%) solution (Prime Dental Products Pvt. Ltd., Thane, India) was used to irrigate the canals during the canal preparation. Nearly 2% chlorhexidine solution (ICPA Health Products Ltd, India) was used as the final irrigant after biomechanical preparation. The root canal treatment was performed in three visits, and calcium hydroxide was used as the intracanal medicament. The root canals were obturated using gutta-percha (Dentsply Maillefer , Ballaigues , Switzerland) and AH 26 sealer (Dentsply DeTrey GmbH, Philadelphia, USA) by lateral compaction technique Before planning for the surgical procedure, patient’s platelet count (4 lakh/mm3 ), hemoglobin (12 g/dl), bleeding time (2.5 min), and clotting time (4.5 min) were assessed and found to be within normal limits. Informed consent was obtained from the patient. Under local anesthesia (1:200,000 adrenaline, DJ Lab, India), a full thickness mucoperiosteal flap was reflected by a sulcular incision starting from the distal aspect of the tooth 12 to distal aspect of the tooth 25 [Figure 2a]. A large periapical defect was seen with complete loss of labial cortical plate

53. . The lesion measured 2.5 cm, 2 cm, and 2 cm corresponding to the length, width, and depth of the lesion. Tissue curettage was done at the defect site followed by thorough irrigation using sterile saline solution [Figure 2b and c]. Using #702 tapered fissure bur (SS White Burs), root end resection was performed in teeth 11, 21, 22, and 23 [Figure 2d]. Root end cavity of 3 mm depth was prepared with diamond-coated ultrasonic surgical tip S12 90ND ( Satelec /Acteon, Merignac, France) at high-power setting of ultrasonic device. White mineral trioxide aggregate (MTA) ( ProRoot MTA; Dentsply, Tulsa, USA) was used as the root end filling material. A volume of 10 mL of blood was drawn from the patient’s antecubital vein and centrifuged (REMI centrifuge machine Model R-8c with 15 mL swing out head) for 10 min under 3000 revolutions

54. 400 g) per minute to obtain the PRF. The resultant product consisted of the following three layers: • A cellular platelet poor plasma at the top of the tube • Fibrin clot (PRF) in the middle of the tube and • Red blood corpuscles at the bottom of the tube. PRF was carried and packed into the defect to the level of defect walls Flap stabilization was done followed by suturing using 3-0 black silk suture material (Sutures India Pvt. Ltd, Karnataka, India). Analgesics and antibiotics were prescribed, and the patient was advised to use 0.2% chlorhexidine mouthwash for a week. Suture removal was done 1 week later and the healing was satisfactory. Patient was reviewed at 3 months [Figure 3a and b] and 12 months [Figure 3c] during which there were no symptoms of pain, inflammation, or discomfort. These follow-up visits included routine intraoral, radiographic examinations, and professional plaque control. Radiographically, periapical bone regeneration was evident at the end of 12 months [

55. Discussion Orthograde root canal therapy should be the first option for treatment of all inflammatory periapical lesions which have 85% of success rate. Periapical surgery remains the last resort when orthograde treatment fails or is not possible. After a surgical procedure, healing usually occurs by repair or regeneration. The four critical factors that influence bone regeneration after the periapical surgery are primary wound closure, angiogenesis as a blood supply and source of undifferentiated mesenchymal cells, space maintenance, and stability of the wound (PASS principle). The present case report evaluated the clinical efficacy of PRF in the treatment of intrabony defect. PRF is a matrix of autologous fibrin with a large quantity of platelet and leukocyte cytokines embedded in it. As the network of fibrin disintegrates, the intrinsic incorporation of cytokines within the fibrin mesh allows their progressive release over time (7–11 days). The main component of PRF is high concentration of growth factors present in the platelets which are required for wound healing.[11-14]

56. Among the various growth factors, PRF contains PDGF, TGF-β1 and β2, IGF, epidermal growth factor (EGF), vascular EGF‑, and fibroblast growth factors which are believed to play a major role in bone metabolism and potential regulation of cell proliferation PDGF is an activator of collagenase which promotes the strength of healed tissue. TGF-β activates fibroblasts to form procollagen which deposits collagen within the wound. PRF facilitates healing by controlling the local inflammatory response. According to Simonpieri et al.,the use of this platelet and immune concentrate during bone grafting offers the following four advantages: First, the fibrin clot plays an important mechanical role and serves as biological connectors between the bone particles. Second, the integration of this fibrin network into the regenerative site facilitates cellular migration, particularly for endothelial cells necessary for the neoangiogenesis , vascularization and survival of the graft. Third, the platelet cytokines (PDGF, TGF-α, IGF-1) are gradually released as the fibrin matrix is resorbed, thus creating a perpetual process of healing. Finally, the presence of leukocytes and cytokines in the fibrin network also plays a significant role in the self‑regulation of inflammatory and infectious phenomena within the grafted material.

57. Conclusion- PRF is a healing biomaterial as it contains all the factors required for optimal wound healing. Previous research and clinical experience indicate that PRF improves early wound closure, maturation of bone, and the final aesthetic result of the periodontal soft tissues. Long-term follow-up of the present case and long-term controlled clinical trials will be required to evaluate the final treatment outcome. Prasanthi NN, Chittem J, Simpsy GS, Sajjan GS. Surgical management of a large inflammatory periapical lesion with platelet-rich fibrin. J Interdiscip Dentistry 2017;7:76-9

58. Within the parameters of this investigation, the following conclusions were drawn: 1. Wound healing responses of the mucoperiosteal tissues to incisional wounding in periradicular surgery are remarkably rapid. 2. Few differences in the temporal and qualitative degrees of healing of incisional wounds were noted between the two types of flap designs, although the submarginal rectangular incisions showed a less predictable healing pattern with greater intersample variations in the first 4 postoperative days. 3. The intrasulcular incision leaves a thin layer of vital tissues attached to supracrestal root surfaces. This root-attached connective tissue and epithelium are not clinically visible. 4. With close flap reapproximation and the formation of a thin fibrin clot in the wound site, apical epithelial downgrowth along the root surface does not occur if the vitality of the root-attached tissues is maintained during and after periradicular surgery. Thus, loss of soft tissue attachment level following periradicular surgery with an intrasulcular incision is not inevitable but is preventable. 5. In the presence of vital root-attached tissues, the temporal and qualitative wound healing in the intrasulcular incisional wound site is essentially the same as that of other incisional wounds evaluated in this study.

59. 6. Vitality of root-attached tissues can be predictably maintained by (a) initiating reflection and elevation of the flap in the vertical incision and using undermining elevation to reflect the flap; (b) avoiding curettement or planing of the supracrestal root surfaces; and (c) preventing the dehydration of these tissues with frequent irrigation. 7. Preservation of root-attached epithelium promotes rapid epithelial seal formation, and preservation of root-attached connective tissue enhances connective tissue reattachment rather than new attachment 8. At 14 and 28 days postsurgery , there is essentially no difference in the incisional wound healing progress of the two flap designs in any of the evaluated or observed biological events of wound healing. 9. In vertical incisional wounds of both flap designs, epithelial closure occurs rapidly, with a multilayered epithelial seal established between 24 and 48 h and epithelial barrier formation occurring between 48 and 72 h. Collagen synthesis in the wound site also occurs early, with aggregation of The Incisional Wound 435 collagen macromolecules to form fibers between 48 and 72 h.

60. . 10. In horizontal wounds of both flaps designs, epithelial closure is extremely rapid; with a thin epithelial seal established at 24 h, a multilayered seal between 48 and 72 h, and epithelial barrier formation occurring between 72 and 96 h. Collagen fibers are formed in the wound site between 24 and 48 h.

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