|Year : 2013 | Volume
| Issue : 1 | Page : 42-50
The diverse roles of soft-tissue grafts in creating perioesthetics
Jaisika Rajpal1, Aakash Arora2, Jagriti Gupta3, Ruchika Prasad4
1 Department of Periodontology, Subharti Dental College, Meerut, India
2 Department of Oral and Maxillofacial Surgery, Subharti Dental College, Meerut, India
3 Department of Oral Pathology, Sardar Patel Dental College, Lucknow, Uttar Pradesh, India
4 Department of Oral Medicine and Diagnosis, Sardar Patel Dental College, Lucknow, Uttar Pradesh, India
|Date of Web Publication||26-Nov-2013|
45/A, Aashirwad Bhawan, Beside Maittri Niwas Guest House, Krishna Nagar, Kanpur Road, Lucknow - 226 023, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
As patients become increasingly aware of the esthetic potential through cosmetic dentistry, there is an increasing demand for smile makeovers with perioplastic surgeries. When gum tissue recedes due to periodontal disease, it pulls away from the teeth. Periodontal plastic surgery procedures can restore some coverage and dramatically improve a person's smile. Soft-tissue grafts and other root coverage procedures cover exposed roots and restore healthy gum tissue. This will reduce further bone loss and recession, make the tooth less sensitive, protect the root from root cavities and look more natural when you smile. In this paper, the scope of periodontal plastic surgery with the use of soft-tissue autografts has been outlined to aid the dental team in the proper diagnosis and treatment of the esthetic dental case.
Keywords: Free gingival graft, papilla reconstruction, perioplastic surgeries, subepithelial connective tissue grafts, width of attached gingiva
|How to cite this article:|
Rajpal J, Arora A, Gupta J, Prasad R. The diverse roles of soft-tissue grafts in creating perioesthetics. Int J Oral Health Sci 2013;3:42-50
| Introduction|| |
The healthy soft-tissue surrounding the natural dentition is composed of the gingiva and alveolar mucosa, which are clearly demarcated into clinically identifiable zones. The free gingiva begins at the gingival margin, which is normally located 1-3 mm coronal to the cemento-enamel junction and extends to the base of the gingival sulcus. The attached gingiva refers to the tissue that is firmly bound by Sharpey's fibers to the cementum of the tooth and underlying bone and begins at the base of the gingival sulcus in health - or periodontal pocket in disease - and extends to the mucogingival junction. The apical migration of the gingival margin results in gingival recession, which may lead to root exposure that not only may be aesthetically unacceptable to the patient, but more importantly, is always accompanied by bone loss. This migration can also result in tooth sensitivity, difficulty in plaque removal, root caries and cervical abrasion and erosion. Gingival recession is a common clinical finding that affects almost 90% of the American population. 
Gingival health can usually be maintained with as little as 1 or 2 mm of attached gingiva. ,, Occasionally, with good oral hygiene, movable alveolar mucosa can be maintained indefinitely. ,,, However, soft-tissue grafting procedures are often indicated to optimize function and esthetics. Clinical situations in which grafting is indicated include the following:
- Areas of minimal or no attached gingiva where the movable alveolar mucosal margins interfere with plaque control. These areas become chronically inflamed and may be further compromised by a frenum pull or shallow vestibule ,
- Areas of progressive recession ,
- Areas in which orthodontic procedures may position the roots of a tooth in a prominent part of the arch, or when a tooth is tipped lingually, resulting in buccal displacement of the roots 
- Areas where there is a need for restorative treatment and the margins of the restorations will be placed subgingivally and impinge upon the biologic width of connective tissue attachment.  It is generally accepted that 5 mm of keratinized gingiva are required prior to restorative procedures (2 mm is the average width of the free gingiva plus approximately 3 mm of attached keratinized gingiva). Soft-tissue grafting procedures may also be indicated if the clasps of a removable prosthesis irritate the marginal tissues 
- Areas where recession presents an esthetic concern to the patient
- Areas where root exposure has resulted in tooth sensitivity. In younger patients who present with any of the factors mentioned above (especially orthodontics), soft-tissue grafting should be considered to help reduce future complications.
Friedman in 1957 defined mucogingival problems to increase the width of attached gingiva and described denudation and pushback procedures for their correction.  In 1963, Bjorn described the first free gingival graft (FGG).  In that study, FGGs were used to gain keratinized attached gingiva. Bjorn's technique to extend the vestibular fornix was presented in the United States by King and Pennel at the Philadelphia Academy of Periodontology in 1964. 
Mucogingival defects can now be corrected by several periodontal plastic surgical techniques, including (1) laterally or coronally advanced pedicle grafts, (2) coronally advanced flaps alone or in conjunction with barrier membranes or enamel matrix proteins, (3) FGGs and (4) connective tissue autografts and allografts. Each technique has its indications, advantages and limitations. However, the amount of root coverage that can be achieved through periodontal plastic surgery can be predicted based upon Miller's classification of marginal tissue recession.  Upon healing, 100% of root coverage is obtained if the marginal tissue is at the CEJ, the sulcus depth is 2 mm or less and there is no bleeding on probing.
| Gingival Grafting Techniques|| |
FGG for root coverage:
1. The FGG is an autograft obtained from a palatal donor site, an edentulous ridge, or tuberosity. After transplantation to the recipient site, the graft benefits from plasmic diffusion from the adjacent tissue. This helps sustain the graft over avascular root surfaces. The graft's connective tissue will determine the surface appearance of the new gingiva. If it is obtained from the palate, the mature graft may resemble palatal tissue, resulting in esthetic complications. ,
The application of a FGG for root coverage was first described by Nabers in 1966 and with few modifications, the principles and techniques described by Sullivan and Atkins in 1968 are still valid. ,,
The FGG has the advantage of being a predictable procedure when properly performed. However, the FGG may result in an unaesthetic "patch-like" appearance and is therefore often contraindicated in the esthetic zone. It is especially indicated for vestibular extension procedures, but the size of the transplanted graft is limited by the availability of donor tissue.
| Case Reports|| |
A 29-year-old male patient presented for a periodontal evaluation prior to orthodontic treatment. Of concern was the gingival recession in the area of tooth No. 31 and 41. The remainder of the sextant had a very thin layer of keratinized attached gingiva that could be susceptible to recession, especially if the roots of the teeth were to be torqued buccally during the orthodontic treatment [Figure 1]. After preparing the recipient bed for the FGG, the donor tissue was harvested from the palate [Figure 2] and [Figure 3]. The raw wound on the palatal donor site was then covered with the healing stent to promote healing and prevent patient discomfort. The FGG was secured using interrupted sutures and periosteal sling sutures. It was ensured that the graft remained immobile and firmly bound to the underlying periosteum [Figure 4]. After 1-year of treatment, there was complete root coverage and maturation of the graft [Figure 5].
|Figure 1: Miller's Class I gingival recession in tooth 31 and Class II gingival recession in tooth 41|
Click here to view
|Figure 2: The incision placed on the palate for the retrieval of free gingival graft|
Click here to view
|Figure 5: The post-operative view after 1 year showing complete coverage|
Click here to view
Free gingival graft for increasing the width of attached gingiva
The keratinized attached gingiva provides the periodontium with increased resistance to external injury, contributes to the stabilization of the gingival margin and aids in dissipating physiological forces exerted by the muscular fibers of the alveolar mucosa on the gingival tissues.  Increasing attached gingiva should be strongly considered in cases where the patient's plaque control is compromised. The apically positioned flap, FGG and subepithelial connective tissue graft (SCTG) are the most common surgical procedures used for augmenting the zone of attached gingiva effectively and predictably. The newly obtained keratinized gingiva can be maintained for a long period; in addition, these periodontal procedures halt the progression of gingival recession and could lead to gaining more keratinized gingiva from creeping attachment after the surgery.
Although the implications of findings from previous studies should be considered when planning periodontal treatment that involves mucogingival surgery, there are some indications for surgical intervention. ,, Mucogingival surgical procedures should be strongly considered when the patient's plaque control is compromised. For teeth with little or no attached gingiva that require prosthetic restorations or orthodontic treatment or have an abnormal frenal attachment, the zone of the attached gingiva must be increased.  Attached gingiva also needs additional width when the pocket depth extends beyond the alveolar mucosa.
A FGG refers to grafting of a piece of gingiva (including the keratinized epithelium and periodontal connective tissue) to the recipient site after it has been detached completely from the donor site.  Prior to the re-establishment of vascularization, the FGG survives by consuming nutrients from the cut blood vessels of the recipient site into the graft. By the second day, the blood supply is re-established in the graft through anastamosis; it continues to mature for the next 28 days. 
During a periodontal examination of a 45-year-old female it was found that there was an inadequate band of the gingiva on the facial aspect of the mandibular incisors [Figure 6]. There was Grade III recession on tooth no. 31 and 41. The teeth also presented with Grade II mobility. There was mild to moderate gingival inflammation marginally. As there was the loss of interdental bone and soft-tissue apical to the cementoenamel junction the decision was taken to augment the attached gingiva, in order to prevent the progression of recession. The FGG from palate was planned to be used for increasing the width of attached gingiva. After anesthetizing the recipient and the donor site, an incision was placed at the level of mucogingival margin and a partial thickness flap was raised and recipient bed prepared in the region of mandibular incisor teeth [Figure 7]. A FGG was then obtained from the palate the graft was trimmed and excess tissue removed to achieve the appropriate thickness. The graft was then sutured and stabilized over the recipient bed [Figure 8]. The graft was further protected using the periodontal dressing. A stent was placed on the palate to aid in uneventful healing by secondary intention. A sufficient amount of stable keratinized tissue and adequate vestibular depth was achieved at the treated site after 6 months [Figure 9].
|Figure 6: Inadequate gingival width and recession in mandibular incisor region|
Click here to view
|Figure 7: Partial thickness flap raised after incision at mucogingival junction for preparation of the recipient site|
Click here to view
|Figure 9: The increase in the width of attached gingival and vestibular depth after 6 months|
Click here to view
2. SCTG for root coverage: The SCTG is one of the most versatile and predictable periodontal plastic surgical procedures. It consists of a bilaminar reconstruction of the gingiva using both free and pedicle connective tissue layers to preserve graft viability over denuded root surfaces.  Because of the dual blood supply to the graft (from the underlying connective tissue and the overlying flap), the SCTG results in improved root coverage.  The results are limited by the amount of avascular root surface and the interdental periodontal attachment levels. Based on Miller's classification, virtually 100% root coverage can be anticipated in Class I and Class II defects where there is no interproximal loss of bone or gingiva, but is limited in Class III and IV defects where there is interdental periodontal attachment loss. 
It has been shown that it is the underlying connective tissue that determines epithelial differentiation.  Therefore, since only the connective tissue is transplanted in the "interpositional" SCTG, the result is an enhanced color match and more esthetic results due to the surface characteristics of the overlaying flap. In addition, because the connective tissue is harvested from beneath a partial thickness flap, wound healing in both the donor and recipient sites occurs mostly by primary intention.
This helps expedite maturation and also reduces post-operative discomfort. SCTGs are not indicated in areas where the surface characterization of the gingival tissue needs to be changed (such as with amalgam tattoos). Furthermore, in patients with very thin palatal tissue, alternate donor sites (such as the retromolar pad) or alternative procedures should be considered. As with all periodontal surgical procedures, the SCTG is technique sensitive. To avoid surgical complications, thorough knowledge of the anatomy of the area is imperative.
A 16-year-old female patient presented with the complaints of temperature sensitivity and aesthetic concerns in the areas of gingival recession. Teeth no. 31 had gingival recession [Figure 10]. Patient did not have any interproximal bone loss, the interdental gingival level was normal and the recession did not extend beyond the mucogingival junction (Miller Class I). Complete root coverage could therefore be anticipated. The recipient site was prepared with a full-partial thickness trapezoidal gingival flap elevation [Figure 11]. The flap was extended well beyond the mucogingival junction so that it exhibited no tension when pulled coronally beyond the cementoenamel junction. The donor connective tissue was harvested from under a flap created in the ipsilateral palate [Figure 12]. The connective tissue graft was placed on saline soaked gauze while the palatal wound was closed. The palatal flap was then re-approximated and sutured for primary intention closure [Figure 13]. The interpositional SCTG was then placed on the recipient site and covered with the gingival flap [Figure 14]. Complete root coverage and enhanced gingival esthetics were obtained after 8 months of maturation [Figure 15].
|Figure 11: Full-partial thickness flap reflected beyond mucogingival junction|
Click here to view
|Figure 14: Connective tissue graft sutured on recipient bed and flap coronally repositioned and sutured over the graft|
Click here to view
3. SCTG for papilla reconstruction: One of the major esthetic challenges in periodontal plastic surgery is related to the ability of rebuilding the lost papilla in the maxillary anterior segment. Absence of the papilla with opening of the black spaces result in "Black triangles" and they are one of the most important aspects of the decision making process of a clinician. This condition may create esthetic impairments and food impactions.  The common causes of the loss of the inter-dental papilla are midline diastema, diverging roots, tooth extractions, traumatic interproximal oral hygiene procedures, abnormal crown forms and periodontal diseases.  Several surgical and non-surgical techniques have been proposed to treat soft-tissue deformities and to manage the inter-proximal space. The non-surgical approaches are orthodontic, prosthetic and restorative procedures. They modify the interproximal space, thereby inducing modifications of the soft-tissues. The surgical techniques aim to recontour, preserve or reconstruct the soft-tissue between the teeth and the implants.  This case report highlights the importance of the inter-proximal papilla and it's clinical significance and the progress of the papilla augmentation, based on the use of a sub-epithelial connective tissue graft for the treatment of the "Black triangle."
A 37-year-old healthy woman who was sought treatment of her shifting maxillary teeth and closure of diastemas [Figure 16]. A diagnosis of generalized advanced chronic periodontitis in the maxillary arch was made. There was no mobility of teeth and no high frenums were associated with the diastema. After the sanative phase I therapy, the papilla reconstruction using connective tissue graft was planned. After anesthetizing, the area a semilunar incision was made 3 mm apical to the mucogingival junction facial to the inter-dental area, followed by a pouch like preparation toward the inter-dental area. Intra-sulcular incisions were made around the necks of the adjacent teeth to free the connective tissue attachment from the root surface, to allow the coronal displacement of the gingivo-papillary unit [Figure 17]. The donor sub-epithelial connective tissue graft, which was harvested from the palate was tucked in and pushed coronally within the prepared pouch to support and provide bulk to the coronally positioned interdental papilla [Figure 18]. The gingivopapillary unit was then sutured and a periodontal dressing was applied. There was an increase of 3 mm in the height of the inter-dental papilla within 6 months and a 100% of papilla fill was obtained [Figure 19].
|Figure 17: Semilunar incision placed 3 mm apical to the mucogingival junction, Intrasulcular incisions given and tunnel prepared|
Click here to view
|Figure 18: Connective tissue graft tucked in the pouch and the gingivo-papillary unit coronally repositioned and sutured|
Click here to view
| Discussion|| |
Depending on the patient's situation, various techniques can be performed to increase attached gingiva effectively. An apically positioned flap should be the first choice when attached gingiva is available. The apically positioned flap has been used for gingival pocket reduction while preserving or increasing keratinized attached gingiva. Studies have proven that apically positioned flap techniques can establish adequate zones of attached gingiva without deforming the dentogingival junction.  Because gingival recession is the major risk from an apically positioned flap, this procedure is recommended primarily when pocket depth exists or when a gingivectomy could remove all or most of the attached gingiva.
One of the most common approaches for gingival augmentation, the FGG procedure has proven to be efficient and predictable for increasing the apico-coronal dimension of attached gingiva.  A 2008 retrospective study reported that FGGs provided long-term (10-25 years) stability.  One potential advantage of FGGs is that post-operative migration of the gingival marginal tissue in a coronal direction may occur and cover a previously denuded root surface partially or even totally; however, such "creeping attachment" occurs unpredictably after the healing of FGGs. Based on the author's experience, FGG procedures could provide better esthetics by avoiding the keloid line between the previous gingiva and the new graft tissue.
Despite these positive aspects, attempts to cover areas of deep, wide gingival recession with FGG procedures were unpredictable for many years.  In 1983, the study by Holbrook and Ochsenbein reported that FGG provided complete root coverage in 22 of 50 cases (44%).  After 10 years, Jahnke et al. reported a mean root coverage of 43% following FGG. Although Miller demonstrated that successful root coverage was possible for Class I and Class II recessions, root coverage was not the immediate and primary goal of FGG procedures. 
Gingival recession related to periodontal disease or developmental problems can result in trapped plaque, root sensitivity, root caries and esthetic compromises. Langer and Langer described a SCTG technique for root coverage in which a partial-thickness flap with two vertical incisions was used to prepare the recipient site. 
Several studies have shown that the SCTG is a predictable procedure for obtaining esthetic root coverage.  A 2002 study reported that the root coverage (measured by reduction of gingival recession) gained from a SCTG was maintained for a long period (mean 27.5 months). There was a statistically significant increase in the mean root coverage between the short-term follow-up (97.1%) and the long-term follow-up (98.4%).  The fact that the mean root coverage improved with time supports the concept of creeping attachment when autogenous soft-tissue grafts are used.  Based on the significant decrease in recession and an increase in the quantity of keratinized tissue over time, the subepithelial CTG is recommended when root coverage is attempted. 
| Conclusion|| |
Most of the studies indicate that with adequate plaque control, minimal to no attached gingiva may be maintained in a state of health over long periods of time. However, there are clear indications for augmenting the zone of keratinized gingiva. Numerous periodontal plastic surgical procedures have been developed to correct mucogingival defects and to increase the zone of keratinized attached gingiva. Three of the commonly used procedures - the FGG, the SCTG and acellular dermal connective tissue allograft procedures - were described. Each procedure has clear advantages and disadvantages that need to be evaluated according to the patient's needs. In addition, all procedures are limited by the amount of avascular root surface, the height of the interproximal papillae and the alveolar bone. Moreover, several mucogingival conditions may occur concurrently, necessitating the consideration of combining or sequencing surgical techniques.
| References|| |
|1.||Miller A, Brunelle J, Carlos J. Oral Health of United States Adults: The National Survey of Oral Health in U.S. Employed Adults and Seniors, 1985-1986: National Findings. Bethesda, Md: US Department of Health and Human Services, Public Health Service; 1987. NIH Publication, 87-2868. |
|2.||Lang NP, Löe H. The relationship between the width of keratinized gingiva and gingival health. J Periodontol 1972;43:623-7. |
|3.||Hangorsky U, Bissada NF. Clinical assessment of free gingival graft effectiveness on the maintenance of periodontal health. J Periodontol 1980;51:274-8. |
|4.||de Trey E, Bernimoulin JP. Influence of free gingival grafts on the health of the marginal gingiva. J Clin Periodontol 1980;7:381-93. |
|5.||Wennström JL. Lack of association between width of attached gingiva and development of soft tissue recession. A 5-year longitudinal study. J Clin Periodontol 1987;14:181-4. |
|6.||Wennström J, Lindhe J. Plaque-induced gingival inflammation in the absence of attached gingiva in dogs. J Clin Periodontol 1983;10:266-76. |
|7.||Salkin LM, Freedman AL, Stein MD, Bassiouny MA. A longitudinal study of untreated mucogingival defects. J Periodontol 1987;58:164-6. |
|8.||Freedman AL, Green K, Salkin LM, Stein MD, Mellado JR. An 18-year longitudinal study of untreated mucogingival defects. J Periodontol 1999;70:1174-6. |
|9.||Gottsegen R. Frenum position and vestibule depth in relation to gingival health. Oral Surg Oral Med Oral Pathol 1954;7:1069-78. |
|10.||Gorman WJ. Prevalence and etiology of gingival recession. J Periodontol 1967;38:316-22. |
|11.||Baker DL, Seymour GJ. The possible pathogenesis of gingival recession. A histological study of induced recession in the rat. J Clin Periodontol 1976;3:208-19. |
|12.||Maynard JG Jr, Ochsenbein C. Mucogingival problems, prevalence and therapy in children. J Periodontol 1975;46:543-52. |
|13.||Ericsson I, Lindhe J. Recession in sites with inadequate width of the keratinized gingiva. An experimental study in the dog. J Clin Periodontol 1984;11:95-103. |
|14.||Maynard JG Jr, Wilson RD. Physiologic dimensions of the periodontium significant to the restorative dentist. J Periodontol 1979;50:170-4. |
|15.||Friedman N. Mucogingival surgery. Tex Dent J 1957;75:358-62. |
|16.||Bjorn H. Free transplantation of gingival propria. Odontol Revy 1963;14:523. |
|17.||King K, Pennel B. Evaluation of attempts to increase the width of attached gingiva. Presented at the Philadelphia Society of Periodontology, 1964. |
|18.||Miller PD Jr. A classification of marginal tissue recession. Int J Periodontics Restorative Dent 1985;5:8-13. |
|19.||Karring T, Lang NP, Loe H. Role of connective tissue in determining epithelial specificity. J Dent Res 1972;51:1303-4. |
|20.||Karring T, Lang NP, Löe H. The role of gingival connective tissue in determining epithelial differentiation. J Periodontal Res 1975;10:1-11. |
|21.||Nabers JM. Free gingival grafts. Periodontics 1966;4:243-5. |
|22.||Sullivan HC, Atkins JH. Free autogenous gingival grafts. I. Principles of successful grafting. Periodontics 1968;6:121-9. |
|23.||Sullivan HC, Atkins JH. Free autogenous gingival grafts. 3. Utilization of grafts in the treatment of gingival recession. Periodontics 1968;6:152-60. |
|24.||Carnio J, Camargo PM, Passanezi E. Increasing the apico-coronal dimension of attached gingiva using the modified apically repositioned flap technique: A case series with a 6-month follow-up. J Periodontol 2007;78:1825-30. |
|25.||Ainamo J, Talari A. The increase with age of the width of attached gingiva. J Periodontal Res 1976;11:182-8. |
|26.||Ainamo A, Ainamo J, Poikkeus R. Continuous widening of the band of attached gingiva from 23 to 65 years of age. J Periodontal Res 1981;16:595-9. |
|27.||Snyder AJ. A technic for free autogenous gingival grafts. J Periodontol 1969;40:702-6. |
|28.||Serio FG, Hawley CE. Lexi-Comp's Manual of Clinical Periodontics: A Reference Guide for Diagnosis and Treatment. 2 nd ed. Hudson, OH: Lexi-Comp; 2002. p. 95. |
|29.||Harris RJ. Root coverage with connective tissue grafts: An evaluation of short-and long-term results. J Periodontol 2002;73:1054-9. |
|30.||Nelson SW. The subpedicle connective tissue graft. A bilaminar reconstructive procedure for the coverage of denuded root surfaces. J Periodontol 1987;58:95-102. |
|31.||Tarnow DP, Magner AW, Fletcher P. The effect of the distance from the contact point to the crest of bone on the presence or absence of the interproximal dental papilla. J Periodontol 1992;63:995-6. |
|32.||Prato GP, Rotundo R, Cortellini P, Tinti C, Azzi R. Interdental papilla management: A review and classification of the therapeutic approaches. Int J Periodontics Restorative Dent 2004;24:246-55. |
|33.||Fagan F. Clinical comparison of the free soft tissue autograft and partial thickness apically positioned flap - Preoperative gingival or mucosal margins. J Periodontol 1975;46:586-95. |
|34.||Dordick B, Coslet JG, Seibert JS. Clinical evaluation of free autogenous gingival grafts placed on alveolar bone. Part II. Coverage of nonpathologic dehiscences and fenestrations. J Periodontol 1976;47:568-73. |
|35.||Agudio G, Nieri M, Rotundo R, Cortellini P, Pini Prato G. Free gingival grafts to increase keratinized tissue: A retrospective long-term evaluation (10 to 25 years) of outcomes. J Periodontol 2008;79:587-94. |
|36.||Matter J. Creeping attachment of free gingival grafts. A five-year follow-up study. J Periodontol 1980;51:681-5. |
|37.||Holbrook T, Ochsenbein C. Complete coverage of the denuded root surface with a one-stage gingival graft. Int J Periodontics Restorative Dent 1983;3:8-27. |
|38.||Miller PD Jr. Root coverage with the free gingival graft. Factors associated with incomplete coverage. J Periodontol 1987;58:674-81. |
|39.||Langer B, Langer L. Subepithelial connective tissue graft technique for root coverage. J Periodontol 1985;56:715-20. |
|40.||Wennström JL. Mucogingival therapy. Ann Periodontol 1996;1:671-701. |
|41.||Cordioli G, Mortarino C, Chierico A, Grusovin MG, Majzoub Z. Comparison of 2 techniques of subepithelial connective tissue graft in the treatment of gingival recessions. J Periodontol 2001;72:1470-6. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15], [Figure 16], [Figure 17], [Figure 18], [Figure 19]