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Methods for Evaluation of Regenerative
Procedures* Samuel E.
A
Lynch
multitude of new procedures and materials are
being tested for their ability to
promote periodontal wound healing. This review provides an overview of the most well-
accepted methods for evaluating periodontal wound healing procedures/materials. Topics discussed include appropriate patient selection, general principles in non-surgical and surgical therapy as they relate to wound healing trials, clinical soft and hard tissue measurements which are generally obtained, an overview of radiographie assessments, and a discussion of the methods and role of histological evaluations in clinical trials. The advantages and disadvantages of the various methodologies are presented. The review concludes with the potential endpoints which can be used in periodontal wound healing studies. / Periodontal 1992; 63:1085-1092. Key Words: Wound healing; periodontal diseases/therapy.
Procedures used to treat periodontal lesions are currently deemed clinically successful if they slow the progression of the destructive process or aid in restoring a portion of one or more components of the periodontium. However, periodontal disease remains the leading cause of tooth loss in adults. Clearly, there is a need for new procedures or materials which promote periodontal wound healing and lead to the restoration of the periodontal attachment apparatus. In this regard, a multitude of new procedures and materials are being tested for their ability to promote periodontal wound healing, the ultimate goal being to develop a method to regenerate the entirety of the lost periodontium. The aim of this paper is to describe the most widely accepted methods for evaluating regenerative procedures. In order to understand the rationale which underlies the methods used to assess periodontal regeneration, a few basic definitions are helpful. Regeneration: The reproduction or reconstitution of a lost or
injured part.1
Periodontal regeneration: Reconstitution of the periodontal attachment apparatus including the gingival attachment, cementum, periodontal ligament, and alveolar bone around teeth with a deficient periodontium. New Attachment: The reunion of connective tissue with a root surface that has been deprived of its periodontal ligament. This formation occurs by the formation of new cementum with inserting collagen fibers.2 Regeneration differentiates from new attachment in one basic, but very important way: alveolar bone formation is a prerequisite in regeneration of the periodontium. Areas of new cementum deposition without adjacent new bone 'Institute of Molecular
Biology, Inc., Worcester,
MA.
and vice versa, and areas of ankylosis, for example, do not satisfy the criteria for regeneration. These definitions are important since some therapeutic modalities may predictably promote new attachment but not bone, while other therapies may promote bone to the exclusion of the periodontal ligament. Neither of these types of therapies would lead to regeneration. The methods for evaluating regenerative procedures also differ substantially from those used to evaluate therapeutics aimed at promoting only new attachment. In general, more thorough evaluation of the treatment results are necessary when documenting regeneration. Primarily this involves not only evaluation of attachment levels but also evaluation of the changes in alveolar bone height. The methods described here are generally equally applicable for evaluation of both the procedures aimed at achieving new attachment and those aimed at achieving regeneration, with the exception that the methods for assessing hard tissue changes are applicable only for procedures aimed at promoting regeneration. Patient Selection General methods for
selecting the appropriate patient popdiscussed elsewhere in this supplement. The specific inclusion and exclusion criteria required for studies evaluating regenerative procedures following adult onset Periodontitis are shown in Table 1. These criteria are meant to provide general guidelines for patient recruitment. They may be modified or other criteria may be added as appropriate for the individual regenerative procedures. ulation
are
and Surgical Therapy The exact nature of the presurgical and surgical will vary widely depending upon the procedure or
Presurgical
therapy product
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EVALUATION OF REGENERATIVE PROCEDURES
to be evaluated. In general, presurgical care should include a comprehensive treatment plan to provide for the patient's
dental needs. Normal standards for dental care should be met. An occlusal analysis and, if warranted, occlusal adjustment should be performed during this period. Although often apparently overlooked, adjustment of the occlusion is critical in wound healing studies to eliminate fremitus and reduce mobility. Regeneration around a tooth in movement due to occlusal forces is difficult, if not impossible. Root planing of study teeth should be avoided if the study's aim is to evaluate the ability of the test procedure/product to promote new attachment on previously diseased root surfaces. Baseline data should be collected during these phases for the methods involved in evaluation of treatment efficacy. The osseous defects should be preliminarily classified by number of bone walls (0 to 4). During the surgical therapy a notch may be placed in the roots of teeth having a hopeless prognosis at the base of the calculus. This notch delineates exposed and non-exposed root surfaces. Notch placement has been reported using a 1/4 round bur with a fiber optic light source and magnifying loops serving as aids to visualize the apical extent of the calculus.3"5 However, despite attempts to the contrary, it is often difficult to visually locate the apical extent of the calculus deposits and accurately place a notch at this location. This is especially true in deep 2- and 3wall osseous defects. A notch may be placed in a similar fashion at the alveolar crest or base of the osseous defect. Clinically, the latter notches can be placed with greater ease, accuracy, and reproducibility. Regardless of their exact location, demarcations at specified locations in the root are necessary to allow subsequent histological evaluation. Following placement of anatomic landmarks, thorough root planing should be performed exercising great care not to obscure the notches, and the osseous defect should receive final classification. The regenerative material is then placed and the gingival flaps adapted closely to the tooth surface. Primary closure is nearly always considered advantageous in regenerative procedures. Methods which have been employed to quantify tissue changes due to putative regenerative techniques or products can be divided into 5 general categories: 1) clinical evaluation of attachment levels and other soft tissue parameters; 2) clinical evaluation of hard tissue changes; 3) radiographic evaluation of hard tissue changes; 4) histological evaluation of biopsy material; and 5) other ancillary methods. Each of these methods will be described and their individual advantages and disadvantages will be discussed. Recommendations for the adoption of a composite of these methodologies when evaluating putative regenerative products will be presented. CLINICAL MEASUREMENTS OF SOFT TISSUE Clinical measurements offer a non-invasive means of evaluating changes in soft tissue adaptation to the root surface. When used in conjunction with other methods they can
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Table 1. General Patient Inclusion and Exclusion Criteria for Regenerative Studies
Inclusion 1. At least 1 tooth in 2 contralateral quadrants with similar osseous defects 2. Angular osseous defects > 4 mm in depth 3. Clinical vertical attachment loss > 6 mm; clinical horizontal attachment loss ?" 4 mm 4. Involved teeth are vital and asymptomatic 5. Patients are systemically healthy 6. Patients are over the age of 35 (or over the age of 21 if juvenile
Periodontitis cases are specifically excluded) give informed consent Patients demonstrate acceptable oral hygiene prior to surgical therapy Patients are willing and able to return for multiple follow-up visits The involved teeth are recommended for extraction due to periodontal or prosthetic considerations by 2 dentists not involved in the study (for histological studies)
7. Patients
8. 9. 10.
Exclusion 1. Lack of appropriate periodontal defects 2. Endodontic involvement of study teeth 3. Mobility of study teeth > Grade II 4. Current systemic diseases known to interfere with wound healing (e.g., chronic liver failure, chronic renal failure, AIDS, alcoholism, collagen vascular diseases, etc.) 5. Taking drugs known to interfere with wound healing (e.g., corticosteroids, chemotherapeutic anti-cancer drugs, immune modulators, etc.) or who have received such drugs within 4 weeks of treatment in study 6. Smoking or other use of tobacco products 7. Inability or unwillingness to give informed consent 8. Unacceptable oral hygiene habits 9. History of juvenile Periodontitis
provide substantive information on the effects of a putative regenerative therapy. Hundreds of articles have utilized this approach to evaluate the effects of almost innumerable substances or procedures intended to slow the progression of periodontal diseases or aid in the healing of a damaged periodontium. Methods The methods for measuring clinical attachment levels have been discussed the paper by Pihlstrom in this supplement (1072-1077). In general all methods require a fixed reference point from which to obtain reproducible measurements. The cemento-enamel junction (CEJ), edge of a stent, root notch, or restoration have all been utilized. The CEJ offers the advantage of being a readily-available natural landmark. However it does not allow for reproducible placement of a probe (or other measuring device) in a mesio-distal location. Thus, if the CEJ is used as a landmark, a detailed protocol for mesio-distal probe placement and angulation must also be established. Another disadvantage of the CEJ is that it may be obscured by soft tissue, particularly at the post-treatment evaluation period. Customized acrylic occlusal Stents with grooves at the desired probing locations offer the advantage of providing a fixed reference point apico-coronally and mesio-distally. If the teeth are mobile, however, stability of the stent can be compromised over time. In this instance a nightguard can help maintain the teeth in their original positions.
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Various types of calibrated probes have been used to obtain the linear measurements. Calibration of probes prior to initiation of the study is essential since significant variability can occur in factory markings. Automated probes can also be used and offer their own advantages and disadvantages, as has been discussed previously. Other instruments used to obtain measurements include calibrated roundtipped endodontic spreaders and endodontic silver points used in conjunction with locking pliers and a Boley gauge. The exact methods of obtaining clinical soft tissue measurements may not be critical as long as their reproducibility has been verified prior to initiation of the study. Whenever possible measurements should be made by the same investigator throughout the study. That is, the investigator who obtains the baseline measurements for a particular set of parameters should, ideally, take the measurements for those same parameters post-treatment. By having measurements taken by the same investigator pre- and posttreatment, inter-examiner variability is eliminated. When it is not possible for one investigator to perform the measurements at all necessary time-intervals, all investigators involved in a particular set of measurements should be carefully standardized prior to initiation of the study. Preferably inter-examiner standardization should be performed during examination of patients with a similar degree of disease as those patients which will be studied. In addition, detailed written guidelines should be provided which clearly specify how and where measurements are to be obtained. The following measurements have been appropriately utilized to evaluate the effects of putative regenerative procedures on soft tissues (Table 2): 1. Height of free gingival margin (FGM). Distance from the landmark (CEJ, edge of stent, notch, etc.) to the coronal extent of gingiva. 2. Probing (clinical) attachment level (PAL/CAL). Vertical distance from the landmark to the base of the periodontal pocket. 3. Probing pocket depth (PD). Vertical distance from the coronal extent of the gingiva to the base of the soft tissue
pocket.
4. Horizontal furcation probing depth (HFPD). Horizontal distance from the base of the pocket within the furcation to inner edge of the free gingiva. 5. Stent to CEJ (S-CEJ). Distance from the edge of the stent to the CEJ (used to check complete seating of the stent). A certain minimum level of disease must be present prior to initiation of the study to allow for a reasonable chance of detecting a significant improvement due to the treatment. In general, vertical probing pocket depths and attachment levels should exceed 5 mm and horizontal furcation probing depths should exceed 3 mm. However, the most important aspect in horizontal furcation probing as it pertains to patient eligibility is not a certain minimum distance but rather the presence of a substantial Class II furcation lesion. The appropriate methods of analyzing data to detect significant changes in measured endpoints obtained in clinical studies
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Table 2. Clinical Soft Tissue Measurements 1. Height of free gingival margin (FGM) 2. Probing (clinical) attachment level (PAL/CAL) 3. Probing pocket depth (PD) 4. Horizontal furcation probing depth (HFPD) 5. Stent to CEJ (S-CEJ)
are
discussed in another paper by
supplement.
Imrey and Chilton in this
Advantages of the Clinical Evaluation of Soft Tissue The major advantages of the clinical evaluation of soft tissue changes are: 1) they can be performed routinely on large numbers of patients; 2) they provide clinically relevant data
which historically has been the basis for determination of tooth prognosis and evaluation of treatment success; and 3)
they are relatively inexpensive to perform.
Disadvantages of the Clinical Evaluation of Soft Tissue The disadvantages of the clinical evaluation of soft tissue
1) the types of tissue attachment cannot be determined; 2) the effect of the therapy on hard tissue cannot be evaluated; and 3) there can be significant inter-examiner and, to a lesser extent, intra-examiner variability if rigorous procedures for standardization are not followed. changes
are:
CLINICAL MEASUREMENTS OF HARD TISSUE The clinical measurement of hard tissues offers a compromise between non-invasive clinical soft tissue measurements and highly invasive histological measurements of soft and hard tissue biopsies. While a second or re-entry surgery is required, it is usually quicker and less traumatic than the original surgery. When used in combination with the evaluation of clinical soft tissue changes, these measurements can provide substantial information regarding the efficacy of therapeutic modalities aimed at regenerating the periodontium. They should be obtained at the time the regenerative procedure is performed and following complete healing, usually 6 to 12 months later. The following linear measurements of hard tissue changes, taken from a fixed reference point, are considered appropriate for evaluating putative regenerative procedures (Table 3): 1. Crestal height of bone (CEJ/S-AC). The distance from the landmark (e.g., CEJ or stent) to the alveolar crest. 2. Bone loss (CEJ/S-BD). The distance from the landmark to the base of the osseous defect. 3. Defect depth (AC-BD). The distance from the alveolar crest to the base of the osseous defect. 4. Open horizontal furcation probing depth (OHFPD). The distance from the buccal or lingual/palatal extent of the osseous furcation defect to the outer aspect of a probe placed level with the fornix of the furcation and connecting the prominences of the adjacent roots. A volumetric assessment of osseous defects may also be useful in certain instances. In general 2 methods have been utilized. In the first method, the osseous defect (either in
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EVALUATION OF REGENERATIVE PROCEDURES
Table 3. Clinical Bone Measurements 1. Crestal height of bone (CEJ/S-AC) 2. Bone loss (CEJ/S-BD) 3. Defect depth (AC-BD) 4. Open horizontal furcation probing depth
5. Furcation defect volume
(FDV)
(OHFPD)
the patient or in a stone model produced from an impression of the defect in the patient) is filled with a material. The amount of material required to fill the defect is subsequently weighed. For example, a vinyl polysiloxane elastomeric impression material has been injected from a syringe into furcation defects.6 After the material set, it was carved with a sharp blade to an imaginary plane level with the prominences of the 2 roots. The material was then removed and weighed. In the second method the volume of the defects was determined by injection of a gelatinous substance into the furcation defect from a 100 µ syringe. The defect volume was determined to be the number of microliters of gel expelled from the syringe in order to fill the defect.7
Advantages of the Clinical Evaluation of Hard Tissue
The clinical evaluation of hard tissues provides a direct 3dimensional measure of the effects of the putative regenerative procedure on bone formation or résorption. In addition, the procedure allows direct visualization of the supracrestal root surface and, if present, the implanted test material. Alterations of these surfaces can thus be readily assessed. While not documented thoroughly, there appears to be less intra- and inter-examiner variability in these measurements than in many other clinical measurements.
Disadvantages of the Clinical Evaluation of Hard Tissue The major disadvantage to the clinical evaluation of hard tissue changes is that it requires a second, or re-entry, surgical procedure. It should also be noted that clinical evaluation of hard tissue responses is only advantageous when a regenerative procedure or product is being tested and not when a procedure or product is aimed at promoting only new attachment. RADIOGRAPHIC ASSESSMENT OF HARD TISSUE The procedure for assessing bone changes radiographically is reviewed in other papers by Jeffcoat and Reddy in this supplement. It is worth emphasizing again, however, that radiography currently provides the only non-invasive method for evaluating changes in hard tissue around teeth. Thorough validation that radiographie methods accurately reflect clinical changes in bone levels and volume of bone defects, for specific defect types, may obviate the need for direct clinical evaluation of bone changes. This would obviously be beneficial as it would eliminate the surgical re-entry procedure. The best results are clearly generated when standardized radiographs are evaluated using computerized digital subtraction radiography techniques. Measurements which can
(Supplement)
be accurately made using these techniques include: 1) changes in bone height both at the osseous crest and at the base of the defect adjacent to the root surface; 2) changes in bone density; and 3) changes in the percentage of bone support for individual roots. HISTOLOGICAL EVALUATION Periodontal regeneration and new attachment can only be truly verified at the microscopic level. When assessing regeneration, this verification requires demonstration of new cementum and periodontal ligament in addition to the coronal growth of alveolar bone; i.e., the formation of a complete new attachment apparatus (CNAA). When assessing new attachment, histologie verification requires the demonstration of only new cementum with inserting collagen fibers. Controversy exists as to whether the formation of new cementum with inserting collagen fibers must occur on previously-diseased root surfaces in order to satisfy the most accepted definition of new attachment. Clearly, demonstration of the components of new attachment on a previously-diseased root surface meets more stringent criteria for success. To determine if new attachment has occurred on a previously-exposed root surface the apical extent of the diseased root must be marked clinically, usually by notching through calculus, at the time of surgery.5'8 While histologie evaluation provides the only true means of determining regeneration or new attachment, it is extremely difficult to obtain biopsies of the teeth and surrounding structures from humans with sufficient predictability and reliability to allow meaningful statistical analysis. However, numerous case studies have reported histologie findings following placement of putative regenerative substances in humans.9"14 Bowers and co-workers have also reported a series of important studies in which they report statistical analysis of human histologie data generated in controlled clinical studies. The latter studies evaluated the healing around nonsubmerged and submerged roots, submerged roots with and without bone grafts, and non-submerged roots with and without bone grafts.3"5,15
Obtaining the Biopsy Specimens
Biopsies after the
of the treated sites
can
be obtained at any time Most studies indicate that
regenerative procedure. cementogenesis has peaked at 3 months.16"18 However, at least 6 months is recommended to allow for the variability in healing between individuals. One to 2 years may be necessary to allow for complete bone formation in sites subjected to guided tissue regeneration procedures. The following method has been described by Bowers and co-workers for obtaining biopsies.4 A horizontal incision is made in the keratinized gingiva coronal to the mucogingival junction, being careful not to disturb the marginal tissue. Vertical incisions are made mesially and distally to the biopsy site. A split thickness flap is then reflected to a level 2 mm apical to the base of the original osseous defect ~
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LYNCH
1089
apical extent of calculus as judged using previous clinical measurements. A number 700 L cross-cut fissure bur in a high speed handpiece is used with constant irrigation to section the bone and tooth root. A small interproximal wedge or
of tissue and a section of root, approximately 5 mm wide, 7 mm long, and 5 mm thick is removed. The lingual/palatal or buccal cortical plate (whichever is opposite the defect), if present, is preserved. The biopsy material is then placed in fresh 10% buffered formalin or other appropriate fixative and transferred to the histopathology laboratory for embedding and sectioning. Step-serial sections (5 to 8 µ in thickness) are then cut in a mesio-distal plane if the original osseous lesion was located interproximally or within a furcation. Alternatively, sections can be made in a buccal-lingual plane if the original lesion was located on the buccal or lingual/palatal surfaces. At least 2 sections at defined intervals should be mounted and stained. One section should be stained with hematoxylin and eosin and the second section may be stained with either a Masson/Mallory/Goldner's trichrome, reticulum, or von Kossa stain, von Kossa staining for mineralized tissues is only performed on non-decalcified specimens.
Measurements The following measurements have been recorded from evaluation of human histological material. Ideally these measurements should be obtained approximately every 500 µ and recorded by 2 examiners unaware of the treatment rendered to the individual sites (Fig. 1, Table 4). 1. Complete new attachment apparatus (CNAA). The distance from the apical extent of the notch at the base of the calculus or defect to the coronal extent of new cementum with adjacent new bone and an intervening periodontal
Figure 1. Histological measurements for evaluation of regenerative pro-
cedures: NAA—New attachment apparatus; NC—New cementum; NB— New bone; AC—Alveolar crest; CT—Connective tissue attachment; JE— Junctional epithelium; A—Notch at clinical location of alveolar crest; B— Notch at base of cakulusldefect; D—Dentin; OC—Old cementum; PL— Periodontal ligament; BP—Bone particle (Reprinted with permission4). Table 4. 1. 2. 3. 4. 5. 6. 7. 8. 9.
Histological Soft and Hard Tissue Measurements
Complete New Attachment Apparatus (CNAA) New Cementum (NC) New Connective Tissue Attachment (CT) New Bone (NB) Initial Defect Depth (B-A) Final Defect Depth (B-Ac) Crestal Resorption (A-Ac) Junctional Epithelium (B-Je) Junctional Epithelium (Ac-Je)
ligament.
2. New cementum (NC). The distance from the apical extent of the notch at the base of the calculus/defect to the coronal extent of new cementum. 3. New connective tissue attachment (CT). The distance from the coronal extent of NC to the apical extent of the
junction epithelium.
4. New bone (NB). The distance from the apical extent of the notch at the base of the calculus/defect to the most coronal aspect of new bone. 5. Initial defect depth (B- ). The distance from the apical extent of the calculus notch or notch at the base of the defect to the apical extent of a notch at the alveolar crest. 6. Final defect depth (B-Ac). The distance from the coronal extent of the CNAA to the crest of alveolar bone upon microscopic examination. 7. Crestal résorption ( -Ac). The distance from the apical extent of the notch at the initial alveolar crest to the crest of alveolar bone upon microscopic examination. 8. Junctional epithelium (B-Je). The distance from the apical extent of the notch at the base of the calculus/defect to the apical extent of the JE. 9. Junctional epithelium (Ac-Je). The distance from the
microscopic alveolar crest to the apical extent of the junctional epithelium. The photomicrographs in Figures 2 through 4 depict the typical appearance of the regenerating periodontium in dogs at 5, 12, and 24 weeks of healing. These sites originally exhibited natural periodontal disease characterized by moderate to severe bone loss, generalized marginal erythema and edema, purulent exúdate, plaque, and calculus. The sites were treated with flap surgery during which the combination of platelet-derived growth factor (PDGF) and insulin-like growth factor-I (IGF-I) (a method previously shown to enhance periodontal wound healing)19'20 was applied to the exposed root surfaces and bone. They illustrate how histology can be used to verify regeneration/CNAA formation as well as many of the other parameters described above.
of Histological Evaluation advantages of histological evaluation are that it is the primary way to provide convincing evidence of true regeneration or new attachment. It also is the primary means available to definitively discern the anatomical relationships
Advantages The
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EVALUATION OF REGENERATIVE PROCEDURES
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Figure 2. Photomicrograph depicting the typical appearance of the regenerating periodontium obtained 5 weeks following the application of the PDGF-BB/IGF-I combination to a natural, class III furcation lesion in a dog. The apical extent of root planing (i.e., original defect depth) is indicated by the arrow. Note the restoration of the bone, periodontal ligament, and cementum in the proper anatomical relationship. At this time the new bone has an immature woven appearance (original magnification x40). of the components of the periodontium (e.g., to discern the location of the apical extent of the junctional epithelium).
Disadvantages of Histological Evaluation
The obvious major disadvantage of histological evaluation is that an invasive procedure is necessary to obtain the biopsy. This makes the routine procurement of human biopsy material unreasonable. In good conscience biopsy material can only predictably be obtained from teeth with a hopeless prognosis. These teeth often have excessive mobility and other factors which make extrapolations to less involved teeth difficult. Careful guidelines must be established by the investigator and Institutional Review Board for Human Studies which accommodate the needs and rights of the patient. Pre-clinical histological data is more readily obtained and should itself provide convincing evidence of the treatment outcomes of the therapeutic modality. Thus, it appears reasonable that pre-clinical histological data can
Figure 3. Photomicrograph depicting the typical appearance of the regenerating periodontium 3 months following the application of PDGFBB/IGF-I to a natural periodontal lesion in a dog. New cementum (curved arrows) is present along nearly the entire root surface. The apical extent of root planing (black on white arrow) is present near the inferior border of the photograph. The dotted line indicates the demarcation between the coronal extent of the original bone (OB) present at the time of the treatment and the newly formed bone (NB). A developing periodontal
ligament is present between the new cementum and new bone resulting in CNAA formation (original magnification x 63).
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LYNCH Table 5.
1091
Ancillary Measurements
1. Gingival index 2. Bleeding upon probing 3. Mobility 4. Plaque index 5. Local complications due to
procedure
described previously. In addition they do not directly assess the effects of the test substance on regeneration or new attachment. However, the gingival index, bleeding upon probing, and mobility may provide an indication of the overall healing response. Thus, if the test substance is thought to effect general wound healing processes, these measurements may provide useful adjunctive information. In addition the latter (i.e., dehiscences and other local complications) is of significant interest to the clinician and the FDA, and thus should be closely monitored. The plaque index and bacterial serotyping are most appropriate if the test substance has antimicrobial actions. However, the plaque index also provides useful information for patient motivational purposes and may perhaps be useful in explaining outlying data. Various methods for assessing changes in host immunologie responses may also be justified if the putative regenerative substance has anti-inflammatory properties or otherwise affects the host immunologie response. SUMMARY In summary there
are 5 major methods used to evaluate regenerative procedures or products: 1 and 2) clinical evaluation of soft and hard tissues changes; 3) radiographie evaluation of hard tissue changes; 4) histological evaluation of biopsy material; and 5) other ancillary methods. Utilizing these methods the recommended endpoints for studies assessing regenerative procedures, as shown in Table 6, are: 1. Qinical soft tissue parameters, including probing pocket depths, probing attachment levels, and degree of gingival
recession. 2. Study Figure 4. Photomicrograph depicting the typical appearance of the regenerating periodontium 6 months following the application ofPDGFBB/IGF-I to a natural periodontal lesion in a dog. The apical extent of the notch placed at the crest of the bone at the time of surgery is indicated by the arrow. Completely mature cortical bone, periodontal ligament and cementum (i.e., CNAA) has been regenerated coronal to the notch (original magnification, x 63). and should be used to substantiate, clinical histological data.
by necessity,
limited
ANCILLARY Various ancillary parameters are often evaluated following the use of putative regenerative substances or procedures (Table 5). These include the gingival index, bleeding upon probing, mobility, plaque index, and dehiscences or other local complications. These ancillary measurements are often less objective or more difficult to reproduce than the measurements
endpoints relating to the clinical hard tissue changes including length of bone gain/loss within defect, length of crestal bone gain/loss, and percent defect filllinear (special circumstances: volumetric). 3. Radiographie endpoints are the length of bone gain/ loss within defect, the length of crestal bone gain/loss, and
the percent of defect fill-linear. 4. Histologically appropriate endpoints include the amount of new bone, the length of new cementum, the length of CNAA, the percent of the defect filled with NB, NC, and CNAA, the length of crestal bone ß / , and the length of apical migration of junctional epithelium. 5. Other ancillary endpoints might include gingival index, mobility, bleeding upon probing, plaque index, dehiscences and other complications specific to the test material. Much of the histological data can be obtained pre-clinically in blinded, controlled animal studies utilizing appropriate models. However, at least a small amount of human histologie data would be necessary to support claims of
J Periodontol 1092 Table 6.
EVALUATION OF REGENERATIVE PROCEDURES
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Summary of Recommended Endpoints
Clinical-Soft Tissue
Clinical-Hard Tissue 1. Probing pocket depths 1. Length of bone gain/loss 2. Probing attachment levels within defect 3. Degree of gingival 2. Length of crestal bone recession gain/loss Percent of defect fill-linear, (special circumstances
1.
Radiographic Length of bone gain/loss within defect
2.
Length of crestal bone gain/loss
3. Percent of defect filllinear
volumetric)
Histological 1. Amount of new bone 2. Length of new cementum 3. Length of CNAA Percent of the defect filled with NB, NC and CNAA Length of crestal bone ré6.
sorption/formation Length of apical migra-
1. 2. 3. 4. 5.
Ancillary Gingival index Mobility Bleeding upon probing Plaque index Dishiscences, and other complications specific to procedure
tion of functional
epithelium
in humans. It is important to note, however, that a therapy may clearly improve the clinical prognosis of a tooth—as judged by clinical and radiographie evaluation—without the prerequisite of histological verification of
regeneration
regeneration. Regeneration of the periodontium is the ultimate goal of any product for treating periodontal diseases. The safety and efficacy of a putative regenerative procedure or product should initially be evaluated in animal studies. The data from these studies should substantiate the safety of the procedure or substance and provide a reasonable indication that it enhances regeneration or new attachment as judged by histological evaluation. Promising procedures must then be
tested in well-controlled human clinical trials. It appears reasonable to first evaluate the clinical soft and hard tissue responses using the methods and endpoints described here. Hard tissue responses may either be evaluated clinically or using validated standardized radiographie techniques. Other parameters specifically evaluating the safety of the test substance also need to be evaluated, but discussion of these is beyond the scope of this paper. If the clinical endpoints clearly indicate that a putative regenerative procedure enhances healing of the soft and hard tissues, limited histologie evaluation of human biopsy material may be justified to demonstrate regeneration of the complete attachment apparatus. Together these methods should provide a thorough and convincing evaluation of the efficacy of putative re-
generative procedures.
7. 8. 9.
10.
11.
12.
13.
14.
15.
16.
17.
REFERENCES 1. The American Academy of Periodontology. Terms. / Periodontol 1986; 56(suppl):25. 2. The American Academy of Periodontology. Terms. J Periodontol 1986; 56(suppl):19.
6.
Glossary of Periodontic Glossary of Periodontic
3. Bowers GM, Chadroff B, Carnevale R, et al. Histologie evaluation of new attachment apparatus formation in humans. Part II. / Periodontol 1989; 60:675-682. 4. Bowers GM, Chadroff B, Carnevale R, et al. Histologie evaluation of new attachment apparatus formation in humans, Part I. / Periodontol 1989; 60:664-674. 5. Bowers GM, Granet M, Stevens M, et al. Histologie evaluation of
18. 19.
20.
new attachment in humans. A preliminary report. J Periodontol 1985; 56:381-396. Pepelassi EM, Bissada NF, Greenwell H, Farah CF. Doxycycline-tricalcium phosphate composite graft facilitates osseous healing in advanced periodontal furcation defects. / Periodontol 1991; 62:106-113. Garrett S, Martin M, Egelberg J. Treatment of periodontal furcation defects. / Clin Periodontol 1990; 17:179-185. Cole RT, Crigger M, Bogle G, et al. Connective tissue regeneration to periodontally diseased teeth. J Periodont Res 1980; 15:1. Saffar JL, Colombier ML, Detienville R. Bone formation in tricalcium phosphate-filled periodontal intrabony lesions. Histological observations in humans. / Periodontol 1990; 61:209-215. Stahl SS, Froum SJ, Tarnow D. Human clinical and histologie responses to the placement of HTR polymer particles in 11 intrabony lesions. / Periodontol 1990; 61:269-274. Stahl SS, Froum SJ, Tarnow D. Human histologie responses to guided tissue regenerative techniques in intrabony lesions. Case reports on 9 sites. / Clin Periodontol 1990; 17:191-198. Stahl SS, Froum SJ. Histologie healing responses in human vertical lesions following the use of osseous allografts and barrier membranes. / Clin Periodontol 1991; 18:149-152. Stahl SS, Froum SJ. Healing of human suprabony lesions treated with guided tissue regeneration and coronally anchored flaps. Case reports. / Clin Periodontol 1991; 18:69-74. Stahl SS, Froum SJ. Histologie evaluation of human intraosseous healing responses to the placement of tricalcium phosphate ceramic implants. J Periodontol 1986: 57:211-217. Bowers GM, Chadroff B, Carnevale R, et al. Histologie evaluation of new attachment apparatus formation in humans, Part III. / Periodontol 1989; 60:683-693. Froum SJ, Thaler R, Scopp IW, Stahl SS. Osseous autografts. II. Histologie response to osseous coagulum-bone blend grafts. J Periodontol 1975; 46:656. Dragoo MR, Sullivan HC. A clinical and histological evaluation of autogenous iliac bone grafts in humans. Part II. External root résorption. / Periodontol 1973; 44:614-625. Dragoo MR. Regeneration of the Periodontal Attachment in Humans. Philadelphia: Lea & Febiger; 1981. Lynch SE, Ruiz de Castilla G, Williams RC, et al. The effects of short-term application of a combination of platelet-derived and insulin-like growth factors on periodontal wound healing. / Periodontol 1991; 62:458-467. Lynch SE, Williams RC, Poison AM, et al. A combination of plateletderived and insulin-like growth factors enhances periodontal regeneration. / Clin Periodontol 1989; 16:545-548.
Send reprint requests to: Dr. Samuel E. Lynch, Institute of Molecular Biology, Inc., One Innovation Dr., Worcester, MA 01605-4308.
Methods for Evaluation of Regenerative
Procedures* Samuel E.
A
Lynch
multitude of new procedures and materials are
being tested for their ability to
promote periodontal wound healing. This review provides an overview of the most well-
accepted methods for evaluating periodontal wound healing procedures/materials. Topics discussed include appropriate patient selection, general principles in non-surgical and surgical therapy as they relate to wound healing trials, clinical soft and hard tissue measurements which are generally obtained, an overview of radiographie assessments, and a discussion of the methods and role of histological evaluations in clinical trials. The advantages and disadvantages of the various methodologies are presented. The review concludes with the potential endpoints which can be used in periodontal wound healing studies. / Periodontal 1992; 63:1085-1092. Key Words: Wound healing; periodontal diseases/therapy.
Procedures used to treat periodontal lesions are currently deemed clinically successful if they slow the progression of the destructive process or aid in restoring a portion of one or more components of the periodontium. However, periodontal disease remains the leading cause of tooth loss in adults. Clearly, there is a need for new procedures or materials which promote periodontal wound healing and lead to the restoration of the periodontal attachment apparatus. In this regard, a multitude of new procedures and materials are being tested for their ability to promote periodontal wound healing, the ultimate goal being to develop a method to regenerate the entirety of the lost periodontium. The aim of this paper is to describe the most widely accepted methods for evaluating regenerative procedures. In order to understand the rationale which underlies the methods used to assess periodontal regeneration, a few basic definitions are helpful. Regeneration: The reproduction or reconstitution of a lost or
injured part.1
Periodontal regeneration: Reconstitution of the periodontal attachment apparatus including the gingival attachment, cementum, periodontal ligament, and alveolar bone around teeth with a deficient periodontium. New Attachment: The reunion of connective tissue with a root surface that has been deprived of its periodontal ligament. This formation occurs by the formation of new cementum with inserting collagen fibers.2 Regeneration differentiates from new attachment in one basic, but very important way: alveolar bone formation is a prerequisite in regeneration of the periodontium. Areas of new cementum deposition without adjacent new bone 'Institute of Molecular
Biology, Inc., Worcester,
MA.
and vice versa, and areas of ankylosis, for example, do not satisfy the criteria for regeneration. These definitions are important since some therapeutic modalities may predictably promote new attachment but not bone, while other therapies may promote bone to the exclusion of the periodontal ligament. Neither of these types of therapies would lead to regeneration. The methods for evaluating regenerative procedures also differ substantially from those used to evaluate therapeutics aimed at promoting only new attachment. In general, more thorough evaluation of the treatment results are necessary when documenting regeneration. Primarily this involves not only evaluation of attachment levels but also evaluation of the changes in alveolar bone height. The methods described here are generally equally applicable for evaluation of both the procedures aimed at achieving new attachment and those aimed at achieving regeneration, with the exception that the methods for assessing hard tissue changes are applicable only for procedures aimed at promoting regeneration. Patient Selection General methods for
selecting the appropriate patient popdiscussed elsewhere in this supplement. The specific inclusion and exclusion criteria required for studies evaluating regenerative procedures following adult onset Periodontitis are shown in Table 1. These criteria are meant to provide general guidelines for patient recruitment. They may be modified or other criteria may be added as appropriate for the individual regenerative procedures. ulation
are
and Surgical Therapy The exact nature of the presurgical and surgical will vary widely depending upon the procedure or
Presurgical
therapy product
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EVALUATION OF REGENERATIVE PROCEDURES
to be evaluated. In general, presurgical care should include a comprehensive treatment plan to provide for the patient's
dental needs. Normal standards for dental care should be met. An occlusal analysis and, if warranted, occlusal adjustment should be performed during this period. Although often apparently overlooked, adjustment of the occlusion is critical in wound healing studies to eliminate fremitus and reduce mobility. Regeneration around a tooth in movement due to occlusal forces is difficult, if not impossible. Root planing of study teeth should be avoided if the study's aim is to evaluate the ability of the test procedure/product to promote new attachment on previously diseased root surfaces. Baseline data should be collected during these phases for the methods involved in evaluation of treatment efficacy. The osseous defects should be preliminarily classified by number of bone walls (0 to 4). During the surgical therapy a notch may be placed in the roots of teeth having a hopeless prognosis at the base of the calculus. This notch delineates exposed and non-exposed root surfaces. Notch placement has been reported using a 1/4 round bur with a fiber optic light source and magnifying loops serving as aids to visualize the apical extent of the calculus.3"5 However, despite attempts to the contrary, it is often difficult to visually locate the apical extent of the calculus deposits and accurately place a notch at this location. This is especially true in deep 2- and 3wall osseous defects. A notch may be placed in a similar fashion at the alveolar crest or base of the osseous defect. Clinically, the latter notches can be placed with greater ease, accuracy, and reproducibility. Regardless of their exact location, demarcations at specified locations in the root are necessary to allow subsequent histological evaluation. Following placement of anatomic landmarks, thorough root planing should be performed exercising great care not to obscure the notches, and the osseous defect should receive final classification. The regenerative material is then placed and the gingival flaps adapted closely to the tooth surface. Primary closure is nearly always considered advantageous in regenerative procedures. Methods which have been employed to quantify tissue changes due to putative regenerative techniques or products can be divided into 5 general categories: 1) clinical evaluation of attachment levels and other soft tissue parameters; 2) clinical evaluation of hard tissue changes; 3) radiographic evaluation of hard tissue changes; 4) histological evaluation of biopsy material; and 5) other ancillary methods. Each of these methods will be described and their individual advantages and disadvantages will be discussed. Recommendations for the adoption of a composite of these methodologies when evaluating putative regenerative products will be presented. CLINICAL MEASUREMENTS OF SOFT TISSUE Clinical measurements offer a non-invasive means of evaluating changes in soft tissue adaptation to the root surface. When used in conjunction with other methods they can
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Table 1. General Patient Inclusion and Exclusion Criteria for Regenerative Studies
Inclusion 1. At least 1 tooth in 2 contralateral quadrants with similar osseous defects 2. Angular osseous defects > 4 mm in depth 3. Clinical vertical attachment loss > 6 mm; clinical horizontal attachment loss ?" 4 mm 4. Involved teeth are vital and asymptomatic 5. Patients are systemically healthy 6. Patients are over the age of 35 (or over the age of 21 if juvenile
Periodontitis cases are specifically excluded) give informed consent Patients demonstrate acceptable oral hygiene prior to surgical therapy Patients are willing and able to return for multiple follow-up visits The involved teeth are recommended for extraction due to periodontal or prosthetic considerations by 2 dentists not involved in the study (for histological studies)
7. Patients
8. 9. 10.
Exclusion 1. Lack of appropriate periodontal defects 2. Endodontic involvement of study teeth 3. Mobility of study teeth > Grade II 4. Current systemic diseases known to interfere with wound healing (e.g., chronic liver failure, chronic renal failure, AIDS, alcoholism, collagen vascular diseases, etc.) 5. Taking drugs known to interfere with wound healing (e.g., corticosteroids, chemotherapeutic anti-cancer drugs, immune modulators, etc.) or who have received such drugs within 4 weeks of treatment in study 6. Smoking or other use of tobacco products 7. Inability or unwillingness to give informed consent 8. Unacceptable oral hygiene habits 9. History of juvenile Periodontitis
provide substantive information on the effects of a putative regenerative therapy. Hundreds of articles have utilized this approach to evaluate the effects of almost innumerable substances or procedures intended to slow the progression of periodontal diseases or aid in the healing of a damaged periodontium. Methods The methods for measuring clinical attachment levels have been discussed the paper by Pihlstrom in this supplement (1072-1077). In general all methods require a fixed reference point from which to obtain reproducible measurements. The cemento-enamel junction (CEJ), edge of a stent, root notch, or restoration have all been utilized. The CEJ offers the advantage of being a readily-available natural landmark. However it does not allow for reproducible placement of a probe (or other measuring device) in a mesio-distal location. Thus, if the CEJ is used as a landmark, a detailed protocol for mesio-distal probe placement and angulation must also be established. Another disadvantage of the CEJ is that it may be obscured by soft tissue, particularly at the post-treatment evaluation period. Customized acrylic occlusal Stents with grooves at the desired probing locations offer the advantage of providing a fixed reference point apico-coronally and mesio-distally. If the teeth are mobile, however, stability of the stent can be compromised over time. In this instance a nightguard can help maintain the teeth in their original positions.
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Various types of calibrated probes have been used to obtain the linear measurements. Calibration of probes prior to initiation of the study is essential since significant variability can occur in factory markings. Automated probes can also be used and offer their own advantages and disadvantages, as has been discussed previously. Other instruments used to obtain measurements include calibrated roundtipped endodontic spreaders and endodontic silver points used in conjunction with locking pliers and a Boley gauge. The exact methods of obtaining clinical soft tissue measurements may not be critical as long as their reproducibility has been verified prior to initiation of the study. Whenever possible measurements should be made by the same investigator throughout the study. That is, the investigator who obtains the baseline measurements for a particular set of parameters should, ideally, take the measurements for those same parameters post-treatment. By having measurements taken by the same investigator pre- and posttreatment, inter-examiner variability is eliminated. When it is not possible for one investigator to perform the measurements at all necessary time-intervals, all investigators involved in a particular set of measurements should be carefully standardized prior to initiation of the study. Preferably inter-examiner standardization should be performed during examination of patients with a similar degree of disease as those patients which will be studied. In addition, detailed written guidelines should be provided which clearly specify how and where measurements are to be obtained. The following measurements have been appropriately utilized to evaluate the effects of putative regenerative procedures on soft tissues (Table 2): 1. Height of free gingival margin (FGM). Distance from the landmark (CEJ, edge of stent, notch, etc.) to the coronal extent of gingiva. 2. Probing (clinical) attachment level (PAL/CAL). Vertical distance from the landmark to the base of the periodontal pocket. 3. Probing pocket depth (PD). Vertical distance from the coronal extent of the gingiva to the base of the soft tissue
pocket.
4. Horizontal furcation probing depth (HFPD). Horizontal distance from the base of the pocket within the furcation to inner edge of the free gingiva. 5. Stent to CEJ (S-CEJ). Distance from the edge of the stent to the CEJ (used to check complete seating of the stent). A certain minimum level of disease must be present prior to initiation of the study to allow for a reasonable chance of detecting a significant improvement due to the treatment. In general, vertical probing pocket depths and attachment levels should exceed 5 mm and horizontal furcation probing depths should exceed 3 mm. However, the most important aspect in horizontal furcation probing as it pertains to patient eligibility is not a certain minimum distance but rather the presence of a substantial Class II furcation lesion. The appropriate methods of analyzing data to detect significant changes in measured endpoints obtained in clinical studies
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Table 2. Clinical Soft Tissue Measurements 1. Height of free gingival margin (FGM) 2. Probing (clinical) attachment level (PAL/CAL) 3. Probing pocket depth (PD) 4. Horizontal furcation probing depth (HFPD) 5. Stent to CEJ (S-CEJ)
are
discussed in another paper by
supplement.
Imrey and Chilton in this
Advantages of the Clinical Evaluation of Soft Tissue The major advantages of the clinical evaluation of soft tissue changes are: 1) they can be performed routinely on large numbers of patients; 2) they provide clinically relevant data
which historically has been the basis for determination of tooth prognosis and evaluation of treatment success; and 3)
they are relatively inexpensive to perform.
Disadvantages of the Clinical Evaluation of Soft Tissue The disadvantages of the clinical evaluation of soft tissue
1) the types of tissue attachment cannot be determined; 2) the effect of the therapy on hard tissue cannot be evaluated; and 3) there can be significant inter-examiner and, to a lesser extent, intra-examiner variability if rigorous procedures for standardization are not followed. changes
are:
CLINICAL MEASUREMENTS OF HARD TISSUE The clinical measurement of hard tissues offers a compromise between non-invasive clinical soft tissue measurements and highly invasive histological measurements of soft and hard tissue biopsies. While a second or re-entry surgery is required, it is usually quicker and less traumatic than the original surgery. When used in combination with the evaluation of clinical soft tissue changes, these measurements can provide substantial information regarding the efficacy of therapeutic modalities aimed at regenerating the periodontium. They should be obtained at the time the regenerative procedure is performed and following complete healing, usually 6 to 12 months later. The following linear measurements of hard tissue changes, taken from a fixed reference point, are considered appropriate for evaluating putative regenerative procedures (Table 3): 1. Crestal height of bone (CEJ/S-AC). The distance from the landmark (e.g., CEJ or stent) to the alveolar crest. 2. Bone loss (CEJ/S-BD). The distance from the landmark to the base of the osseous defect. 3. Defect depth (AC-BD). The distance from the alveolar crest to the base of the osseous defect. 4. Open horizontal furcation probing depth (OHFPD). The distance from the buccal or lingual/palatal extent of the osseous furcation defect to the outer aspect of a probe placed level with the fornix of the furcation and connecting the prominences of the adjacent roots. A volumetric assessment of osseous defects may also be useful in certain instances. In general 2 methods have been utilized. In the first method, the osseous defect (either in
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EVALUATION OF REGENERATIVE PROCEDURES
Table 3. Clinical Bone Measurements 1. Crestal height of bone (CEJ/S-AC) 2. Bone loss (CEJ/S-BD) 3. Defect depth (AC-BD) 4. Open horizontal furcation probing depth
5. Furcation defect volume
(FDV)
(OHFPD)
the patient or in a stone model produced from an impression of the defect in the patient) is filled with a material. The amount of material required to fill the defect is subsequently weighed. For example, a vinyl polysiloxane elastomeric impression material has been injected from a syringe into furcation defects.6 After the material set, it was carved with a sharp blade to an imaginary plane level with the prominences of the 2 roots. The material was then removed and weighed. In the second method the volume of the defects was determined by injection of a gelatinous substance into the furcation defect from a 100 µ syringe. The defect volume was determined to be the number of microliters of gel expelled from the syringe in order to fill the defect.7
Advantages of the Clinical Evaluation of Hard Tissue
The clinical evaluation of hard tissues provides a direct 3dimensional measure of the effects of the putative regenerative procedure on bone formation or résorption. In addition, the procedure allows direct visualization of the supracrestal root surface and, if present, the implanted test material. Alterations of these surfaces can thus be readily assessed. While not documented thoroughly, there appears to be less intra- and inter-examiner variability in these measurements than in many other clinical measurements.
Disadvantages of the Clinical Evaluation of Hard Tissue The major disadvantage to the clinical evaluation of hard tissue changes is that it requires a second, or re-entry, surgical procedure. It should also be noted that clinical evaluation of hard tissue responses is only advantageous when a regenerative procedure or product is being tested and not when a procedure or product is aimed at promoting only new attachment. RADIOGRAPHIC ASSESSMENT OF HARD TISSUE The procedure for assessing bone changes radiographically is reviewed in other papers by Jeffcoat and Reddy in this supplement. It is worth emphasizing again, however, that radiography currently provides the only non-invasive method for evaluating changes in hard tissue around teeth. Thorough validation that radiographie methods accurately reflect clinical changes in bone levels and volume of bone defects, for specific defect types, may obviate the need for direct clinical evaluation of bone changes. This would obviously be beneficial as it would eliminate the surgical re-entry procedure. The best results are clearly generated when standardized radiographs are evaluated using computerized digital subtraction radiography techniques. Measurements which can
(Supplement)
be accurately made using these techniques include: 1) changes in bone height both at the osseous crest and at the base of the defect adjacent to the root surface; 2) changes in bone density; and 3) changes in the percentage of bone support for individual roots. HISTOLOGICAL EVALUATION Periodontal regeneration and new attachment can only be truly verified at the microscopic level. When assessing regeneration, this verification requires demonstration of new cementum and periodontal ligament in addition to the coronal growth of alveolar bone; i.e., the formation of a complete new attachment apparatus (CNAA). When assessing new attachment, histologie verification requires the demonstration of only new cementum with inserting collagen fibers. Controversy exists as to whether the formation of new cementum with inserting collagen fibers must occur on previously-diseased root surfaces in order to satisfy the most accepted definition of new attachment. Clearly, demonstration of the components of new attachment on a previously-diseased root surface meets more stringent criteria for success. To determine if new attachment has occurred on a previously-exposed root surface the apical extent of the diseased root must be marked clinically, usually by notching through calculus, at the time of surgery.5'8 While histologie evaluation provides the only true means of determining regeneration or new attachment, it is extremely difficult to obtain biopsies of the teeth and surrounding structures from humans with sufficient predictability and reliability to allow meaningful statistical analysis. However, numerous case studies have reported histologie findings following placement of putative regenerative substances in humans.9"14 Bowers and co-workers have also reported a series of important studies in which they report statistical analysis of human histologie data generated in controlled clinical studies. The latter studies evaluated the healing around nonsubmerged and submerged roots, submerged roots with and without bone grafts, and non-submerged roots with and without bone grafts.3"5,15
Obtaining the Biopsy Specimens
Biopsies after the
of the treated sites
can
be obtained at any time Most studies indicate that
regenerative procedure. cementogenesis has peaked at 3 months.16"18 However, at least 6 months is recommended to allow for the variability in healing between individuals. One to 2 years may be necessary to allow for complete bone formation in sites subjected to guided tissue regeneration procedures. The following method has been described by Bowers and co-workers for obtaining biopsies.4 A horizontal incision is made in the keratinized gingiva coronal to the mucogingival junction, being careful not to disturb the marginal tissue. Vertical incisions are made mesially and distally to the biopsy site. A split thickness flap is then reflected to a level 2 mm apical to the base of the original osseous defect ~
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1089
apical extent of calculus as judged using previous clinical measurements. A number 700 L cross-cut fissure bur in a high speed handpiece is used with constant irrigation to section the bone and tooth root. A small interproximal wedge or
of tissue and a section of root, approximately 5 mm wide, 7 mm long, and 5 mm thick is removed. The lingual/palatal or buccal cortical plate (whichever is opposite the defect), if present, is preserved. The biopsy material is then placed in fresh 10% buffered formalin or other appropriate fixative and transferred to the histopathology laboratory for embedding and sectioning. Step-serial sections (5 to 8 µ in thickness) are then cut in a mesio-distal plane if the original osseous lesion was located interproximally or within a furcation. Alternatively, sections can be made in a buccal-lingual plane if the original lesion was located on the buccal or lingual/palatal surfaces. At least 2 sections at defined intervals should be mounted and stained. One section should be stained with hematoxylin and eosin and the second section may be stained with either a Masson/Mallory/Goldner's trichrome, reticulum, or von Kossa stain, von Kossa staining for mineralized tissues is only performed on non-decalcified specimens.
Measurements The following measurements have been recorded from evaluation of human histological material. Ideally these measurements should be obtained approximately every 500 µ and recorded by 2 examiners unaware of the treatment rendered to the individual sites (Fig. 1, Table 4). 1. Complete new attachment apparatus (CNAA). The distance from the apical extent of the notch at the base of the calculus or defect to the coronal extent of new cementum with adjacent new bone and an intervening periodontal
Figure 1. Histological measurements for evaluation of regenerative pro-
cedures: NAA—New attachment apparatus; NC—New cementum; NB— New bone; AC—Alveolar crest; CT—Connective tissue attachment; JE— Junctional epithelium; A—Notch at clinical location of alveolar crest; B— Notch at base of cakulusldefect; D—Dentin; OC—Old cementum; PL— Periodontal ligament; BP—Bone particle (Reprinted with permission4). Table 4. 1. 2. 3. 4. 5. 6. 7. 8. 9.
Histological Soft and Hard Tissue Measurements
Complete New Attachment Apparatus (CNAA) New Cementum (NC) New Connective Tissue Attachment (CT) New Bone (NB) Initial Defect Depth (B-A) Final Defect Depth (B-Ac) Crestal Resorption (A-Ac) Junctional Epithelium (B-Je) Junctional Epithelium (Ac-Je)
ligament.
2. New cementum (NC). The distance from the apical extent of the notch at the base of the calculus/defect to the coronal extent of new cementum. 3. New connective tissue attachment (CT). The distance from the coronal extent of NC to the apical extent of the
junction epithelium.
4. New bone (NB). The distance from the apical extent of the notch at the base of the calculus/defect to the most coronal aspect of new bone. 5. Initial defect depth (B- ). The distance from the apical extent of the calculus notch or notch at the base of the defect to the apical extent of a notch at the alveolar crest. 6. Final defect depth (B-Ac). The distance from the coronal extent of the CNAA to the crest of alveolar bone upon microscopic examination. 7. Crestal résorption ( -Ac). The distance from the apical extent of the notch at the initial alveolar crest to the crest of alveolar bone upon microscopic examination. 8. Junctional epithelium (B-Je). The distance from the apical extent of the notch at the base of the calculus/defect to the apical extent of the JE. 9. Junctional epithelium (Ac-Je). The distance from the
microscopic alveolar crest to the apical extent of the junctional epithelium. The photomicrographs in Figures 2 through 4 depict the typical appearance of the regenerating periodontium in dogs at 5, 12, and 24 weeks of healing. These sites originally exhibited natural periodontal disease characterized by moderate to severe bone loss, generalized marginal erythema and edema, purulent exúdate, plaque, and calculus. The sites were treated with flap surgery during which the combination of platelet-derived growth factor (PDGF) and insulin-like growth factor-I (IGF-I) (a method previously shown to enhance periodontal wound healing)19'20 was applied to the exposed root surfaces and bone. They illustrate how histology can be used to verify regeneration/CNAA formation as well as many of the other parameters described above.
of Histological Evaluation advantages of histological evaluation are that it is the primary way to provide convincing evidence of true regeneration or new attachment. It also is the primary means available to definitively discern the anatomical relationships
Advantages The
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Figure 2. Photomicrograph depicting the typical appearance of the regenerating periodontium obtained 5 weeks following the application of the PDGF-BB/IGF-I combination to a natural, class III furcation lesion in a dog. The apical extent of root planing (i.e., original defect depth) is indicated by the arrow. Note the restoration of the bone, periodontal ligament, and cementum in the proper anatomical relationship. At this time the new bone has an immature woven appearance (original magnification x40). of the components of the periodontium (e.g., to discern the location of the apical extent of the junctional epithelium).
Disadvantages of Histological Evaluation
The obvious major disadvantage of histological evaluation is that an invasive procedure is necessary to obtain the biopsy. This makes the routine procurement of human biopsy material unreasonable. In good conscience biopsy material can only predictably be obtained from teeth with a hopeless prognosis. These teeth often have excessive mobility and other factors which make extrapolations to less involved teeth difficult. Careful guidelines must be established by the investigator and Institutional Review Board for Human Studies which accommodate the needs and rights of the patient. Pre-clinical histological data is more readily obtained and should itself provide convincing evidence of the treatment outcomes of the therapeutic modality. Thus, it appears reasonable that pre-clinical histological data can
Figure 3. Photomicrograph depicting the typical appearance of the regenerating periodontium 3 months following the application of PDGFBB/IGF-I to a natural periodontal lesion in a dog. New cementum (curved arrows) is present along nearly the entire root surface. The apical extent of root planing (black on white arrow) is present near the inferior border of the photograph. The dotted line indicates the demarcation between the coronal extent of the original bone (OB) present at the time of the treatment and the newly formed bone (NB). A developing periodontal
ligament is present between the new cementum and new bone resulting in CNAA formation (original magnification x 63).
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LYNCH Table 5.
1091
Ancillary Measurements
1. Gingival index 2. Bleeding upon probing 3. Mobility 4. Plaque index 5. Local complications due to
procedure
described previously. In addition they do not directly assess the effects of the test substance on regeneration or new attachment. However, the gingival index, bleeding upon probing, and mobility may provide an indication of the overall healing response. Thus, if the test substance is thought to effect general wound healing processes, these measurements may provide useful adjunctive information. In addition the latter (i.e., dehiscences and other local complications) is of significant interest to the clinician and the FDA, and thus should be closely monitored. The plaque index and bacterial serotyping are most appropriate if the test substance has antimicrobial actions. However, the plaque index also provides useful information for patient motivational purposes and may perhaps be useful in explaining outlying data. Various methods for assessing changes in host immunologie responses may also be justified if the putative regenerative substance has anti-inflammatory properties or otherwise affects the host immunologie response. SUMMARY In summary there
are 5 major methods used to evaluate regenerative procedures or products: 1 and 2) clinical evaluation of soft and hard tissues changes; 3) radiographie evaluation of hard tissue changes; 4) histological evaluation of biopsy material; and 5) other ancillary methods. Utilizing these methods the recommended endpoints for studies assessing regenerative procedures, as shown in Table 6, are: 1. Qinical soft tissue parameters, including probing pocket depths, probing attachment levels, and degree of gingival
recession. 2. Study Figure 4. Photomicrograph depicting the typical appearance of the regenerating periodontium 6 months following the application ofPDGFBB/IGF-I to a natural periodontal lesion in a dog. The apical extent of the notch placed at the crest of the bone at the time of surgery is indicated by the arrow. Completely mature cortical bone, periodontal ligament and cementum (i.e., CNAA) has been regenerated coronal to the notch (original magnification, x 63). and should be used to substantiate, clinical histological data.
by necessity,
limited
ANCILLARY Various ancillary parameters are often evaluated following the use of putative regenerative substances or procedures (Table 5). These include the gingival index, bleeding upon probing, mobility, plaque index, and dehiscences or other local complications. These ancillary measurements are often less objective or more difficult to reproduce than the measurements
endpoints relating to the clinical hard tissue changes including length of bone gain/loss within defect, length of crestal bone gain/loss, and percent defect filllinear (special circumstances: volumetric). 3. Radiographie endpoints are the length of bone gain/ loss within defect, the length of crestal bone gain/loss, and
the percent of defect fill-linear. 4. Histologically appropriate endpoints include the amount of new bone, the length of new cementum, the length of CNAA, the percent of the defect filled with NB, NC, and CNAA, the length of crestal bone ß / , and the length of apical migration of junctional epithelium. 5. Other ancillary endpoints might include gingival index, mobility, bleeding upon probing, plaque index, dehiscences and other complications specific to the test material. Much of the histological data can be obtained pre-clinically in blinded, controlled animal studies utilizing appropriate models. However, at least a small amount of human histologie data would be necessary to support claims of
J Periodontol 1092 Table 6.
EVALUATION OF REGENERATIVE PROCEDURES
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Summary of Recommended Endpoints
Clinical-Soft Tissue
Clinical-Hard Tissue 1. Probing pocket depths 1. Length of bone gain/loss 2. Probing attachment levels within defect 3. Degree of gingival 2. Length of crestal bone recession gain/loss Percent of defect fill-linear, (special circumstances
1.
Radiographic Length of bone gain/loss within defect
2.
Length of crestal bone gain/loss
3. Percent of defect filllinear
volumetric)
Histological 1. Amount of new bone 2. Length of new cementum 3. Length of CNAA Percent of the defect filled with NB, NC and CNAA Length of crestal bone ré6.
sorption/formation Length of apical migra-
1. 2. 3. 4. 5.
Ancillary Gingival index Mobility Bleeding upon probing Plaque index Dishiscences, and other complications specific to procedure
tion of functional
epithelium
in humans. It is important to note, however, that a therapy may clearly improve the clinical prognosis of a tooth—as judged by clinical and radiographie evaluation—without the prerequisite of histological verification of
regeneration
regeneration. Regeneration of the periodontium is the ultimate goal of any product for treating periodontal diseases. The safety and efficacy of a putative regenerative procedure or product should initially be evaluated in animal studies. The data from these studies should substantiate the safety of the procedure or substance and provide a reasonable indication that it enhances regeneration or new attachment as judged by histological evaluation. Promising procedures must then be
tested in well-controlled human clinical trials. It appears reasonable to first evaluate the clinical soft and hard tissue responses using the methods and endpoints described here. Hard tissue responses may either be evaluated clinically or using validated standardized radiographie techniques. Other parameters specifically evaluating the safety of the test substance also need to be evaluated, but discussion of these is beyond the scope of this paper. If the clinical endpoints clearly indicate that a putative regenerative procedure enhances healing of the soft and hard tissues, limited histologie evaluation of human biopsy material may be justified to demonstrate regeneration of the complete attachment apparatus. Together these methods should provide a thorough and convincing evaluation of the efficacy of putative re-
generative procedures.
7. 8. 9.
10.
11.
12.
13.
14.
15.
16.
17.
REFERENCES 1. The American Academy of Periodontology. Terms. / Periodontol 1986; 56(suppl):25. 2. The American Academy of Periodontology. Terms. J Periodontol 1986; 56(suppl):19.
6.
Glossary of Periodontic Glossary of Periodontic
3. Bowers GM, Chadroff B, Carnevale R, et al. Histologie evaluation of new attachment apparatus formation in humans. Part II. / Periodontol 1989; 60:675-682. 4. Bowers GM, Chadroff B, Carnevale R, et al. Histologie evaluation of new attachment apparatus formation in humans, Part I. / Periodontol 1989; 60:664-674. 5. Bowers GM, Granet M, Stevens M, et al. Histologie evaluation of
18. 19.
20.
new attachment in humans. A preliminary report. J Periodontol 1985; 56:381-396. Pepelassi EM, Bissada NF, Greenwell H, Farah CF. Doxycycline-tricalcium phosphate composite graft facilitates osseous healing in advanced periodontal furcation defects. / Periodontol 1991; 62:106-113. Garrett S, Martin M, Egelberg J. Treatment of periodontal furcation defects. / Clin Periodontol 1990; 17:179-185. Cole RT, Crigger M, Bogle G, et al. Connective tissue regeneration to periodontally diseased teeth. J Periodont Res 1980; 15:1. Saffar JL, Colombier ML, Detienville R. Bone formation in tricalcium phosphate-filled periodontal intrabony lesions. Histological observations in humans. / Periodontol 1990; 61:209-215. Stahl SS, Froum SJ, Tarnow D. Human clinical and histologie responses to the placement of HTR polymer particles in 11 intrabony lesions. / Periodontol 1990; 61:269-274. Stahl SS, Froum SJ, Tarnow D. Human histologie responses to guided tissue regenerative techniques in intrabony lesions. Case reports on 9 sites. / Clin Periodontol 1990; 17:191-198. Stahl SS, Froum SJ. Histologie healing responses in human vertical lesions following the use of osseous allografts and barrier membranes. / Clin Periodontol 1991; 18:149-152. Stahl SS, Froum SJ. Healing of human suprabony lesions treated with guided tissue regeneration and coronally anchored flaps. Case reports. / Clin Periodontol 1991; 18:69-74. Stahl SS, Froum SJ. Histologie evaluation of human intraosseous healing responses to the placement of tricalcium phosphate ceramic implants. J Periodontol 1986: 57:211-217. Bowers GM, Chadroff B, Carnevale R, et al. Histologie evaluation of new attachment apparatus formation in humans, Part III. / Periodontol 1989; 60:683-693. Froum SJ, Thaler R, Scopp IW, Stahl SS. Osseous autografts. II. Histologie response to osseous coagulum-bone blend grafts. J Periodontol 1975; 46:656. Dragoo MR, Sullivan HC. A clinical and histological evaluation of autogenous iliac bone grafts in humans. Part II. External root résorption. / Periodontol 1973; 44:614-625. Dragoo MR. Regeneration of the Periodontal Attachment in Humans. Philadelphia: Lea & Febiger; 1981. Lynch SE, Ruiz de Castilla G, Williams RC, et al. The effects of short-term application of a combination of platelet-derived and insulin-like growth factors on periodontal wound healing. / Periodontol 1991; 62:458-467. Lynch SE, Williams RC, Poison AM, et al. A combination of plateletderived and insulin-like growth factors enhances periodontal regeneration. / Clin Periodontol 1989; 16:545-548.
Send reprint requests to: Dr. Samuel E. Lynch, Institute of Molecular Biology, Inc., One Innovation Dr., Worcester, MA 01605-4308.
