12.8 Regeneration of Keratinized Gingiva 12

12.8 Regeneration of Keratinized Gingiva

Michael K. McGuire

12.8.1 Overview The augmentation of keratinized gingiva (KG) around teeth with mucogingival defects has been an achievable goal in dentistry for many years. Compared with the more elastic and unattached alveolar mucosa, “tougher” and attached KG is thought to improve the long-term prognosis for Fig 12.8-1 The (MGJ) can clearly teeth, particularly in patients with questionable be seen in this patient. The probe extends to the MGJ, (Lang and Löe, 1972; Wennström et indicating that this patient has no attached gingiva. al., 1981). The distinct change between kerati- nized, attached gingiva and non-keratinized and tial healing, transplanted graft “shrinkage” loosely attached alveolar mucosa is delineated by occurred, but the resultant dimensions could be the mucogingival junction (MCJ) (Fig 12.8-1) and anticipated and maintained long-term. The sub- distinguished histologically by the distribution and epithelial connective tissue graft (CTG) was also nature of elastic (mucosal) and inelastic (gingival) investigated as an onlay graft (Edel, 1974; Calura collagen fibers and the short, wide (mucosal) and long, slender (gingival) papillae, with a defining layer of keratin on the surface of KG epithelium (Fig 12.8-2). In the 1960s, denudation and pushback pro- cedures were early attempts at increasing the Free gingiva amount of KG, but the outcomes were unpre- Nonelastic connective dictable and painful (Costich and Ramfjord, tissue 1968; Karring et al., 1975). The apically pos- itioned flap (APF) encouraged KG regeneration from the wound bed and margins and, when Keratinized gingiva indicated, was combined with a vestibuloplasty (APF + V) to expose more of the alveolus and Attached gingiva remove the mechanically disruptive influence of muscle pull (Friedman 1962; Robinson and Agnew, 1963). Soft tissue grafting investigations in the 1960s demonstrated that it was possible to Elastic increase KG through the connective tissue Mucogingival (Bjorn, 1963; Pennel, 1969). Studies also indi- junction cated that tissue specificity was retained. Gingi- Alveolar mucosa val grafts harvested from the keratinized gingiva or and transplanted into the mucosa, for example, retained their keratinized and inelastic Fig 12.8-2 Diagram of the adjacent to determination (Karring et al., 1971). During ini- the tooth.

277 Clinical Research Protocols for Indications in Dental Regeneration

In the late 1970s and early 1980s, there were some favorable reports of freeze-dried skin as a substitute for the FGG, but the biomaterial was never widely adopted (Yukna et al., 1977). Cadaveric tissue resurfaced in the late 1990s, when acellular dermal matrix (ADM) was intro- duced as an FGG substitute. Because ADM does not perform well when placed over open wound beds for regenerating KG, the periodontal litera- ture regarding ADM focuses on root coverage procedures with ADM beneath a CAF (Harris, Fig 12.8-3 Postoperative photograph of the 2001; Wei et al., 2000). appearance of a free gingival graft facial to the cuspid Starting in 2003, researchers began reporting and bicuspids. cases in which the patients’ own fibroblasts were cultured and implanted for gingival augmenta- et al., 1991), with the advantage that the CTG tion. In a series of investigations from 2005 to could be harvested via a pouch procedure and 2011, expanded fibroblast and expanded, bilay- might be less uncomfortable postoperatively for ered fibroblast plus keratinocyte (BL) sheets were patients. The regeneration of keratinized and studied (McGuire et al., 2008). Though the attached gingiva appeared to be influenced by amount of KG generated with these tissue-engi- the tissue phenotype of the subepithelial, trans- neered biomaterials was significantly less than planted connective tissue, as well as the remain- the traditional APF + V + FGG standard, over ing periodontal ligament. Accordingly, CTG from 95% of patients in a BL multi center study attached palatal gingiva was placed beneath cor- (McGuire et al., 2007) obtained ≥ 2 mm bands onally advanced flaps (CAF) to accomplish both of KG, and in all of the studies, patients preferred root coverage and improved KG width. However, the esthetic outcome and overall therapy alter- when root coverage was not indicated and a sig- native provided by the tissue engineered solu- nificant amount of KG was desired, the gingival tions. autograft and particularly the free gingival graft (FGG) were adopted as the standard of treat- 12.8.2 Research Question ment (Bjorn, 1963; Nabers, 1966; Sullivan and Though there are abundant systematic reviews Atkins, 1968; Pennell et al., 1969). concerning gingival augmentation procedures for Despite the predictability of the FGG, the tissue root coverage, there are limited reviews concern- texture and color mismatch of the healed trans- ing KG augmentation studies on teeth not requir- plant, sometimes referred to as a “tire patch,” was ing root coverage. A recent and thorough review less than ideal (Fig 12.8-3). Also, complications, (Thoma et al., 2009) underscores that the combi- such as postoperative bleeding and pain coupled nation of APF + V with autogenous graft yields with the finite supply of harvestable donor tissue, the most favorable KG and attached gingiva (AG) prompted researchers to look for substitute bio- results. More randomized controlled trials (RCTs) materials (Griffin et al., 2006). Though regenera- comparing alternative therapies are needed, but tion of a functional zone of KG continued to be with suitable standardization and calibration, the overriding goal, investigators turned to tech- more patient-reported outcomes (PROs), and niques and biomaterials that might minimize mor- adequate statistical analyses and analytical meth- bidity and create tissue indistinguishable from ods, especially with regards to thickness measures, what nature provides. biopsies and histological analyses. The primary

278 12.8 Regeneration of Keratinized Gingiva 12 outcome variable of these KG studies is usually Inclusion change in KG, but secondary outcomes including • Teeth indicated for KG regeneration. change in AG and periodontal health measures • At least two non-adjacent treatment sites of should also be followed. New metrics comparing 1 to 4 teeth span, with <2 mm KG. alternative therapies – for example, surgery time • 18 to 70 years of age. required and PROs measures for anxiety, pain and • (if female) A negative urine test for pregnancy preference – should be compared with the bench- and practicing birth control. mark APF + V + FGG therapy. Ideally, the regen- • Able to read and understand informed consent. erated KG should be indistinguishable from what • Able and willing to follow instructions. nature provides. • Demonstrate good plaque control. • If patients have parafunctional habits, fit with 12.8.3 Time Points bite guard. Six months has been a standard endpoint for most • For PROs – able and willing to participate in gingival augmentation studies, and meta-analyses phone interviews or employ other, “real time” have adopted this endpoint, perhaps by default, monitoring methodologies. as a comparator metric. Early autogenous graft transplant primate research indicated that the Exclusion amount of KG gained at 1 month following initial • Systemic healing conditions.* shrinkage is maintained long term (Karring et al., • Acute infections in the area intended for treat- 1971). In the author’s experience (McGuire et al., ment. 2005, 2008 and 2011), KG width measures • History of tobacco use within past 6 months. observed at 3 months with APF + V + FGG ther- • Intravenous or intramuscular bisphosphonates. apy are maintained to 6 months and beyond. The • Hypersensitivity to any biomaterials to be stud- tissue specifics, i.e., attachment and durability of ied. KG regenerated by new test biomaterials and tis- • Simultaneous participation in other clinical tri- sue-engineered alternatives, however, are still als. unknown. Though the attachment and durability • Previous grafting in the area intended for treat- of the regenerated KG may be initially assessed at ment. 6 months to match historical study metrics, it will • Class V restorations (instability measurement be important, when possible, to design studies landmark). that permit long-term follow-up for 3 to 5 years • Mobility greater than Miller Class II. (Table 12.8-1). • Subjects who, for any reason in the opinion of the investigator, will not be able to complete the 12.8.4 Inclusion and Exclusion Criteria study (e.g., it is not uncommon for patients to The following table provides inclusion and exclu- find out about the study and want to partici- sion guidelines for patients in a contralateral, pate, even though they may not live in the city within-patient, controlled clinical trial. In essence, where the study is being conducted. Also, the systemic health and/or compliance issues should be avoided, while normally encountered, prac- tice-based conditions (e.g., the inclusion of both * Patients with diabetes mellitus, cancer, HIV and/or bone metabolic diseases that could compromise wound healing maxillary and mandibular teeth) should be or preclude periodontal surgery; or who are currently embraced. These latter conditions can sometimes receiving or have received within 2 months prior to study be teased from the results during statistical analy- entry, systemic corticosteroids, immunosuppressive agents, radiation therapy, and/or chemotherapy, which sis to examine whether they impact study end- could compromise wound healing and preclude perio- points. dontal surgery.

279 Clinical Research Protocols for Indications in Dental Regeneration X X X X X X X X X X Post-op Years 1–5 Years ±1 month follow-up Long-term X X X X X X X X X X X Post-op 6 Month Month 6 ± 14 days X X X X X X X Post-op 3 Month Month 3 ± 14 days X X X X X 4 Week 4 Week 4 Week Post-op ± 7 days X X X 1 Week 1 Week 1 Week Post-op ± 3 days X X X X Day 0 / Surgery days of screening Surgery within 30 † X X X X X X X X X X Baseline † surgery X X X X X X X X Within 30 days of Within Screen Visit schedule Typical study timeline. Typical

Study procedures Informed consent Eligibility Demographic Medical history Dental history Oral exam Pregnancy test (if needed) Pregnancy Hygiene procedures Randomization Surgery for surgeries Time Plaque score pocket depth Probing Recession depth Clinical attachment level Muscle pull resistance Inflammation score Keratinized tissue width* Radiographs Table 12.8-1 Table

280 12.8 Regeneration of Keratinized Gingiva 12

patient’s job requirements may not be flexible enough to stay within the allowable windows ‡ X X X X X

X** for study visits).

12.8.5 Endpoints – Primary and Secondary Primary: ∆KGw is the apico-coronal width change ‡ X X X X X

X** in KG, compared with baseline. KGw should be measured with a calibrated from the free gingival margin to the mucogingival junction (MGJ) on the mid-facial, mesial, and dis- ‡ X X X X X

X** tal. If the MGJ is not distinct, the mucosa can be rolled toward the tooth to identify the mucogingi- val junction (Figs 12.8-4a and 12.8-4b). Schiller’s iodine solution (Schiller, 1933) may also be used ‡ X X X X X

X** to demarcate KG and delineate the MGJ transition between keratinized gingiva and non-keratinized mucosa reference. ‡ X X X X Box 12.8-1

It is sometimes difficult to identify the MGJ in photographs. Tissue stained with the iodine solution can help. ‡ X X X X Secondary: Periodontal health (compared with baseline) – change in clinical attachment level (∆CAL), ∆PD, change in recession (∆Rec), change in bleeding upon probing (∆BOP), resistance to muscle pull (the lip or cheek adjacent to the graft X X X X** should be placed under tension to make certain that the treatment area is free of movement dur- ing muscle traction, esthetics (texture and color

X X match compared to surrounding tissue) and inflammation. Inflammation can be graded on a scale such as that depicted in Table 12.8-2. Note that all apico-coronal width measures should be made from a fixed reference point, usu- ally the mid buccal cementoenamel junction (CEJ) of a designated test tooth. When measured by probe, results should be recorded to the nearest 0.5 mm, rounding down and using a calibrated probe such as a University of North Carolina 15 (Hu Friedy, Chicago, IL, USA) with no probing measures made before 3 months to allow uninter- Identification of study teeth Photographs Dental cleaning Texture and color match Texture Subject PROs questionnaire (if needed) Bite guard Medication Adverse events

† The screen and baseline visits may be combined into a single visit. † The screen iodine solution. using Schiller’s of keratinized tissue (mid-facial, mesial, and distal) should be measured * Width iodine solution. ** Photos should be taken with and without Schiller’s the study. ‡ PROs should be collected at various timepoints throughout rupted healing.

281 Clinical Research Protocols for Indications in Dental Regeneration

Fig 12.8-4 (a) A probe can be used to roll the mucosa coronally to help delineate the MGJ. (b) A probe demon- strating freely movable mucosa adjacent to the molar. Note the bleeding and inflammation present.

Table 12.8-2 Inflammation levels and indications. + V + FGG therapy versus the native texture and color of some alternative therapies may be obvi- Score Indication ous to the examiner. Regardless, a “blinded” 0 Normal (absence of inflammation) study should always employ a calibrated examiner 1 Mild inflammation of any portion of separate from the surgeon. the marginal unit (e.g., slight changes in color) Box 12.8-3 2 Mild inflammation of the entire gingival unit (but no edema) A 3 mm biopsy punch is used to take the biopsy. It is initiated at the base of the graft attempting 3 Moderate inflammation (moderate glazing, redness, edema and/or hypertrophy) to bisect the mucogingival junction, so half of the biopsy is mucosa, and half is KG so the MGJ 4 Severe inflammation (marked redness and edema/hypertrophy, spontaneous can be identified in the biopsy. bleeding, or ulceration) When comparing FGGs to new test materials, the examiner may find it useful to biopsy a few of the test and control grafts at 6 months to allow for Box 12.8-2 a blind analysis of the type of tissue generated. If the test graft contains live, allograft cell therapy, Periodontal probes specially calibrated for persistence testing can provide important infor- studies are available. mation regarding both the fate of the transplanted cells as well as safety issues. Biologic graft alterna- A blinded and calibrated examiner, where possi- tives (growth factors and live cell devices, for ble, should make all measurements and assess- example) may also require periodic blood serum ments. However, the realities of “blinding” an testing to demonstrate safety. It is likely that in the examiner to the differences between two thera- future many test grafts will fall into this category, pies can be difficult, if not impossible. For exam- and safety components of the protocol will ple, the “tire patch” appearance of a control APF increase and become more complex.

282 12.8 Regeneration of Keratinized Gingiva 12

Maximum Discomfort at Harvest Site

Mucograft Control ** ** ** ** ** ** ** ** ** ** Maximum Discomfort

Fig 12.8-5 Sample timeline 1 2 3 4 5 6 7 8 9 10 of maximum discomfort at Day *p<.05, **p<.01 the harvest site.

12.8.6 Additional Observations and of “well-defined and reliable” (U.S. Department Experimental Endpoints of Health and Human Services Food and Drug Though the traditional endpoints for a KG regener- Administration: Guidance for Industry 2009). In ation study have been KGw and associated perio- essence, the method and instruments used should dontal health metrics, additional observations and measure what they are intended to measure (reli- exploratory endpoints can also be elucidating. ability), be sensitive to change (able to measure Though unrelated to traditional endpoints, addi- treatment effectiveness differences), and should tional observations may help evaluate study out- be interpretable (clinically meaningful). comes. Exploratory endpoints are measures whose Even though the FDA is mandating that all relationship to established endpoints may be uncer- future studies in the United States collect PROs, tain or unknown at the start of a study and may the ability to glean meaningful information can involve new or untested measurement techniques. be challenging. As an example of the difficulties A simple example of an additional observation in of establishing reliable, effective and clinically KG regeneration studies is the time needed to com- meaningful PROs methodologies for KG regener- plete alternative therapies. For example, compared ation studies, the author is involved in an ongoing with traditional APF + V + autogenous graft ther- study examining a xenogeneic collagen matrix as apy, alternative test therapy with a xenogeneic col- an alternative to autogenous graft harvest for KG lagen matrix has been shown to reduce surgery regeneration. In the course of this investigation, time by over 15 mins on average (Lorenzo et al., peak pain was discovered to occur over the first 2012) – a potential benefit for patient and clinician. few days following therapy (Fig 12.8-5), but the Though therapy time is a relatively simple metric to threshold time point required calling patients evaluate, exploratory PROs endpoints are more dif- daily using 3rd party, uninvolved recorders. ficult, primarily because they are subjective; i.e., Patients may be reluctant to provide unguarded they involve a wide spectrum of patients’ opinions answers when speaking with – and trying to and experiential responses to anxiety, pain/discom- please – their caregiver/staff and surgeon (Orne, fort, treatment preferences and esthetics. 1962). PROs like pain/discomfort must be In 2009, the United States Food and Drug recorded in real time and not when convenient or Administration released guidance about how according to scheduled clinic visits. Statistical PROs should be evaluated to meet the standard analyses must not only account for patient pain

283 Clinical Research Protocols for Indications in Dental Regeneration

Maximum Discomfort (At Treatment and Harvest Site)

Mucograft Control ** ** * ** * * ** Maximum Discomfort

1 2 3 4 5 6 7 8 9 10 Fig 12.8-6 Sample timeline of maximum discomfort (at Day *p<.05, **p<.01 treatment and harvest site).

tolerance differences but also real-time pain still poorly understood and experimental, and why experienced during normal patient activities, such they must be thoroughly vetted and validated as eating, which can involve peak pain experi- before being considered reliable and meaningful. ences brought on by hot, cold or hard foods. Researchers considering PROs measures should refer to the guidelines provided by this text and Box 12.8-4 consider the most recent literature before design- ing and incorporating PROs methodologies in KG Pain is referred to non-operated sites; i.e., the regeneration studies (Newton and Buck DJ, 2000; side of the palate from which no graft is taken Kloostra et al., 2007; Fardal and McCulloch, is also experienced as painful by some patients. 2012; McGrath et al., 2012). PROs are especially important in studies involving alternatives to free These pain experiences will vary between . The universal success rate of the patients because of patient behavior differences, FGG presents a challenge to investigators search- like avoidance of the surgical site when chewing or ing for an alternative therapy that will generate the dulling of pain through the use of discretionary more KG. Alternative endpoints, such as PROs, medication. In the aforementioned study, the may be needed to identify and evaluate therapies author also discovered that surgical pain is referred for patients that are more beneficial (less discom- to non-operated sites; i.e., the side of the palate fort, fewer surgeries, better esthetics, etc.). from which no graft is taken is also experienced as painful by some patients (Fig 12-8-6). Clinical 12.8.7 Test Groups experience indicates that there is pain associated Recent KG regeneration study test alternatives to with the donor site, but it is very difficult to docu- autogenous graft therapy may require special study ment. Very few studies have found a significant design considerations. Autogenous grafts, after ini- difference in pain from harvest versus non-harvest tial shrinkage, tend to retain KGw dimensions soft tissue augmentation therapies, perhaps related to the initial harvest graft width and thick- because the non-harvest, test therapy may require ness; i.e., wider grafts provide more KGw (Mor- a larger recipient bed or more extensive suturing. mann et al., 1981). Therefore, the graft and corre- These examples illustrate why PROs measures are sponding recipient wound bed dimensions must be

284 12.8 Regeneration of Keratinized Gingiva 12 standardized to provide a consistent comparative without the wound dressing that would normally outcome. In the author’s early studies, the width of be employed with an autogenous graft. These the test graft and FGG were always standardized to measures were taken in order to avoid compres- allow for direct comparison of test vs control. How- sion or disruption of the xenogeneic collagen ever, as we learn more about alternative materials, matrix volume – a matrix into which healing tissue it may not be appropriate to standardize the width ingrowth was encouraged. A thorough under- of the grafts. If our goal is to achieve ≥ 2 mm of KT standing (and sometimes pilot clinical work) and we know the test material experiences signifi- should be in hand before designing test group cant shrinkage or degradation after application, it clinical study methodology and beginning a study. may be appropriate to apply 7 to 9 mm of test material (or as much as the depth of the vestibule Box 12.8-5 will allow) in order to achieve or surpass the desired outcome. The need for the increased width can be Surgical technique learning curves should be part of the alternative graft’s handling properties considered for new therapies, and the study and should not be considered a negative for the may benefit from pilot patient procedures fol- material (other than possible increase in pain lowed, but not reported, in results statistics. related to a larger wound bed). On the other hand, it would not be ethical to increase the width If conducting a within-patient therapy compar- (increasing the patient’s chance for pain and bleed- ison, target teeth within contralateral quadrants of ing) of the FGG to match the test material width, the same jaw should be selected. The selection of because we know we can create a functional zone the two target teeth should be relatively matched of KG with a FGG of around 4 mm in width. For in terms of clinical assessment, condition, and these reasons, matching the width of the test and location (i.e., contralateral first bicuspids, con- control grafts may not always be appropriate. Like- tralateral second bicuspids, etc.). Multiple teeth wise, the peculiarities, special handling, surgical can be selected for treatment. When two or more technique differences and unique actions of alter- teeth are selected, the study tooth should be iden- native decellularized allografts, xenografts and tis- tified prior to surgery; when three teeth are sue-engineered, live cell therapies must be consid- selected, the study tooth should be the middle ered in the study design. For example, live cell BL tooth. The number of teeth included in the test sheets, though morphologically and metabolically and control grafts must be identical. The following similar to human skin, are not true grafts, as they selection criteria can be used as a guide when are neither vascularized nor incorporated into selecting target teeth: native healing tissues. BL acts as a healing stimula- • Relatively matched in overall condition. tor, upregulating cytokines and delivering matrix • Relatively matched in terms of recession depth. proteins in synchronization with the requirements • Relatively matched in terms of bleeding. of the healing wound (McGuire et al., 2008). The • Relatively matched in width of keratinized tis- action of BL, therefore, must be considered not as a sue. graft but as a biologic device. • Relatively matched in probing pocket depth. During the investigation, BL was applied in a • Relatively matched in clinical attachment level. z-fold to improve handling over larger wound beds and to deliver the appropriate “dose” of the Before the study is initiated, the protocol and the device necessary to create >2 mm of KT. Similarly, informed consent should be approved by an a xenogeneic collagen matrix, when tested by the appropriate Institutional Review Board (IRB). author in comparison with autogenous graft ther- Signed informed consents should be obtained apy, was applied over a larger wound bed and prior to study participation.

285 Clinical Research Protocols for Indications in Dental Regeneration

12.8.8 Preoperative Care prevent the surgeon from knowing the next treat- Patient medical and dental histories should be col- ment assignment. lected, as well as demographic data (age, gender and ethnicity) and current medications (adminis- Box 12.8-6 tered within the previous 2 months). An oral exam of face, lymph nodes, lips, buccal mucosa, floor of Our preference is to remove all existing KG from mouth, tongue, hard and soft , gingiva and the mucosal flap to better standardize the sites edentulous ridges and teeth should be conducted. A and assure that all KG generated originates from dental cleaning should be performed, and baseline the graft and not from residual KG. clinical measurements obtained. Photographs should be taken, and oral hygiene procedures reviewed. A At surgery, measurements – CEJ to alveolar bone pregnancy test should be performed on women of level, CEJ to surgical position of the coronal and api- childbearing potential. As parafunction can be a cal extent of the graft – should be obtained. Photos confounding factor, patients exhibiting signs of should be taken before, after bed preparation, after clenching or bruxism should be fitted for a bite graft placement, and after dressing is applied, if guard. Periapical radiographs of the study site should applicable. Incision technique should be consistent. be obtained using a bite plane/paralleling device. For example, the coronal sulcular incision should be made at the height of the existing mucosa extend- 12.8.9 Surgical Procedure ing at least to the line angle of the adjacent teeth. (and Randomization) Also a consideration should be made whether to After meeting entry criteria, if study design includes split the band of existing, native KG, so that the comparative therapies, each patient should be entire wound margin might contain KG cell pheno- assigned a sequential identification number corre- type to repopulate the matrix. Vertical incisions can sponding with the order of enrollment. If a with- be made on both the mesial and distal aspects of the in-patient contralateral comparison is employed, study site extending apically as the vestibule allows. the study tooth sites should also be randomly If a decision is made to eliminate all existing KG, the selected. To ensure that there will be no bias in mesial and distal incisions are then connected api- assigning treatment, a predetermined comput- cally and the tissue is discarded. Any muscle fibers er-generated randomization scheme can be used, should be removed to create a clean periosteal bed. taking into account that if palatal graft harvesting is Some studies suggest that a full-thickness horizontal necessary for only one of the therapies, that graft incision should be made just apical, if feasible, to the harvest side is also alternated, i.e., not always planned level of graft placement, separating the matched with the same treatment side for a given periosteum and ensuring that there is no muscle therapy. Each of these factors can be randomized in tension on the bed. The lip or cheek adjacent to the a straight 1:1 ratio without blocking. Sealed enve- graft should be placed under tension to make cer- lopes may be used to identify patient and treat- tain that the treatment area is free of movement ment orders and can be matched with unique and during muscle traction. Placement of surgical dress- sequential patient identification numbers. The ran- ings should be considered in the study design. domization envelopes should remain sealed until the time of surgery and should be opened in order of subject sequence. The opened randomization Box 12.8-7 envelope and insert should be kept with the sub- “Batch surgeries” – for consistency and con- ject’s case report form. Treatment assignments for venience, study surgeries should be scheduled dropouts and ineligible subjects should not be recy- in batches. cled, and a variable block size should be used to

286 12.8 Regeneration of Keratinized Gingiva 12

a b c d

e f g

Fig 12.8-7 (a) Preoperative view of cuspid with recession and an inadequate zone of attached gingiva. (b) Preoperative photograph with iodine stain to help delineate kerati- nized tissue from mucosa. (c) Intraoperative photograph of recipient bed. (d) Intraoperative photograph of free gingival graft sutured to recipient bed. (e) Intraoperative view of palatal donor site. (f) A 6-months postoperative photograph of free gingival graft. Note the increased amount of keratinized tissue present. (g) A 6-months postoperative photograph of free gingival graft after being stained with iodine solution. Note the clear delineation of the MGJ.

Schedule patients and devote particular days for The following are instructions for photograph- studies. Surgical technique and clinical staff per- ing the target areas: formance tend to be more uniform and efficient 1. Take a photograph of the subject initials, subject when performed in sequence rather than sporadi- ID, tooth number, visit number and date. cally. 2. Ensure the site is plaque free and dry by blotting area with gauze. 12.8.10 Study Photographs 3. Photograph must include the incisal edge of the Photographs of each treatment site should be target tooth, and the entire graft must be in the taken before surgery, at baseline or Day 0 visit. photo. On the day of surgery (Day 0), photographs 4. Photograph must include tissue that is 2 mm should be taken of the two prepared graft beds apical to the mucogingival junction. and of the two placed grafts. Photographs of the 5. Photograph must include a least one tooth palatal graft site should also be taken. During sub- adjacent to target tooth on either side. sequent study visits, photographs of each treat- 6. Consistent magnification should be used at 1:1 ment should be obtained. In addition, at Week 1 a and 1:1.5. photograph of the palatal donor site should be 7. Take photos at baseline and at 6 months, with and obtained (Figs 12.8-7a to 12.8-7g). without Schiller’s iodine stain to help identify MGJ.

287 Clinical Research Protocols for Indications in Dental Regeneration

12.8.11 Postoperative Care control to have up to 0.5 mm superiority in the A (0.12%) mouth rinse can be pre- population), μo = hypothesized mean difference = scribed for 30 s twice daily for the first 4 weeks 0.0 mm, σ = the standard deviation of the paired following surgery. Patients should be instructed to mean differences of test and control for the gen- avoid aggressive rinsing and muscle tractioning, or eration of KG from baseline to 6 months = trauma to the treated areas for the first 4 weeks, 1.0 mm, δ = non-inferiority margin = 1.0 mm, α = and should also be instructed not to brush the 0.05, one-sided, 1-β = 0.80. Under these assump- grafted area for the first 2 weeks. If pain/discom- tions, PROC POWER (SAS 9.2; Cary, NC) requires fort PROs measures are recorded, the use of anti- a sample size of 27 evaluable subjects, with a septics and analgesics must be noted. Patients 10% lost-to-follow-up rate, which implies a sam- should be instructed to avoid disruptive (crunchy or ple size of 30 subjects. Therefore, a sample size of sharp) foods for the first 4 weeks following surgery. 30 subjects can be sufficient to meet the primary Blended and other soft foods should be suggested. objective of this type of study. After 2 weeks, subjects may be instructed in a brushing technique using a soft postoperative Box 12.8-8 brush, creating minimal apically directed trauma to the soft tissue of the treated tooth. At 4 weeks, A common problem is insufficient power to subjects can be instructed to stop use of the chlor- evaluate outcomes. Never start a study with- hexidine rinse and resume normal tooth brushing, out first performing sample size calculations gradually resuming interproximal cleaning, as well that assure an adequate level of statistical as chewing, in the treated areas. Following surgery, power. Note that the design choices for the no subgingival instrumentation or probing should study have a significant impact on the required be performed in the experimental areas until 3 sample size. For example, studies that incorpo- months after surgery. Instructions on postoperative rate concepts such as non-inferiority tests, care should be repeated at each appointment. In composite or multiple endpoints, or unpaired particular, the following points should be covered comparisons will significantly alter the sample during the first month: (i) continue chlorhexidine size required to power a study. In addition, spe- rinses; (ii) avoid brushing the graft sites for the first cialized statistical software is required for con- 2 weeks; and (iii) avoid the use of interdental clean- ducting sample size calculations. For these rea- ing devices, chewing on, or trauma to the treated sons, consulting a biostatistician for assistance areas for the first 4 weeks. prior to the onset of the study is recommended.

12.8.12 Sample Size Given a contralateral comparison, within-patient 12.8.13 Measurement of Protocol Specific study design, sample size is predicated on assuring Endpoints (Special Considerations) that the primary endpoint objective has adequate and patients may have different expecta- power to assess the study hypothesis. For a pri- tions regarding KG regeneration therapy out- mary hypothesis that evaluates whether a test is comes. Professionally evaluated outcomes tend to not inferior to control in the generation of KG be more stringent than patient-evaluated out- from baseline to 6 months, sample size can be cal- comes (Kokich et al., 1999). In private practice, culated using a paired t test or non-inferiority test, however, improved PROs are the goal; while sci- such that: H0: μd ≤ μo – δ and Ha: μd > μo - δ, entists may argue over the relative statistical value where: μd = the mean of paired mean differences of one procedure over another, in practice-based of test and control for the generation of KG from settings, treatment success relates more to patient baseline to 6 months = -0.5 mm (i.e., allowing for satisfaction than it does to “millimeters.” “Nearly

288 References 12 as effective” can be an important qualifier if it instruments reliably and sensitively measure clini- takes into consideration PROs outcomes critical to cally meaningful endpoints. PROs must be col- patients. Researchers should expect KG studies to lected in “real time,” and researchers should con- increasingly center on autogenous graft harvest sider the most recent and validated PROs’ alternatives and to incorporate patient expecta- techniques in their study designs. Professionally tions and esthetic goals – emphasizing the goal of evaluated outcomes tend to be more stringent providing regenerated KG indistinguishable from than patient-evaluated outcomes, and patient the native tissue provided by nature. preferences, particularly for less morbid surgeries and more esthetic outcomes, are the focus of the 12.8.14 Conclusion/Summary future. If regeneration of keratinized gingiva is our As clinicians we hope that the regeneration of KG oral health goal, then our therapies must provide will be linked with improved long-term tooth prog- outcomes indistinguishable from what nature pro- nosis. The gingival autograft, and particularly the vides in health, while our treatments must accom- free gingival graft, has become a standard of treat- plish this natural result with the least morbidity ment with predictable KG results, but investigators and the best predictability. are turning to new techniques and biomaterials to minimize morbidity and create tissue indistinguish- Acknowledgments able from what nature provides. Some of these This work was supported, in part, by an educa- therapies, especially those involving live cells or tional grant provided by Geistlich Pharma. Addi- biologics, require novel surgical techniques and tional research referenced in this sub-chapter was analytical methods. A thorough understanding funded by Straumann USA and Organogenesis. (and sometimes pilot clinical work) of a new ther- The authors wish to thank Rebecca Garcia, RDH, apy must be in hand before a study is designed. Director of Clinical Research, Perio Health Clinical Though KG regeneration results can be initially Research Center, Houston, Texas (PHCRC), for assessed at 6 months to match historical study recording outcome measures and coordinating metrics, studies should be designed to permit data, and Cindy Wainscott, CDA, PHCRC for long-term follow-up to 3 to 5 years. Patient inclu- study administration. Special thanks to Todd sion and exclusion criteria should take into account Scantlebury (The Avenues Company, Flagstaff, systemic health and compliance issues, and prac- AZ, USA) for his help with the organization and tice-based patient and treatment realities should production of this manuscript. The authors declare be embraced. Primary KG width outcome should no conflicts of interest. be coupled with traditional periodontal health measures, but PROs are increasingly recognized as important for both researchers and regulators. References Experimental PRO endpoints may more accurately 1. Bjorn H. Free transplantation of gingiva propria. Odon- identify therapies with less discomfort and better tol Revy 1963;14:323. esthetics for patients. 2. Calura G, Mariani G, Parma-Benfenati S, De Paoli S, In general there is a need for more randomized, Lucchesi C, Fugazzatto P. Ultrastructural observations on wound healing of free connective tissue autografts blinded and controlled trials. Suitable standardiza- with and without epithelium in humans. Int J Periodon- tion, calibration, adequate statistical analyses, and tics Restorative Dent 1991;11:65–70. analytical methods should be embraced, and 3. Costich ER, Ramfjord SP. Healing after partial denuda- biopsies with histological analyses incorporated tion of the . J Periodontol 1968;39: 127–134. for more definitive analyses of regenerative 4. Edel A. Clinical evaluation of free connective tissue results. Overall, emphasis must be placed on more grafts used to increase the width of keratinised gingiva. PROs outcomes, in which PROs’ method and J Clin Periodontol 1974;1:185–196.

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