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Tony Melcher, a highly influential and forward-thinking scientist and teacher, focussed on the origins, behaviour and regulation of cells in periodontal tissues. His recent death in April 2020, has motivated us to highlight his multi-level contributions to research in biology and the dental sciences. Tony was particularly adept at recognizing the inherent instructive power of the periodontium, most notably as a model system for studying the inter-relationships between the structure, development and functions of connective tissues. Further, his mentoring of dozens of students who subsequently went on to develop their own careers in research, and his leadership in promoting collaborations in dental sciences world-wide, engendered important advances in the importance and utility of research relating to oral tissues. Here, we reflect upon his development of a large, multi-disciplinary research enterprise, the MRC Group in Periodontal Physiology at the University of Toronto and brief commentaries of those who worked with him there. We examine his early career development and then go on to consider some of his most highly cited publications and their impact on subsequent research trends.

Background Defective cellular elements constitute an important challenge to achieve predictable periodontal regeneration. In an attempt to improve the cellularity of periodontal defects, gingival fibroblasts were implanted without their associated extracellular elements in periodontal defects to expose them to periodontal tissue mediators. In order to investigate the regenerative potential of gingival fibroblasts translocated into periodontal defects, the present study was designed to clinically and biochemically investigate the use of gingival fibroblasts (GF) and their associated mesenchymal stem cells (GMSC) in the treatment of intrabony periodontal defects. Methods A total of 20 subjects were randomly divided into two groups (n = 20). Group I: ten patients were included with ten intrabony periodontal defects that received β-calcium triphosphate (β-TCP) followed by collagen membrane defect coverage, while group II: (10 patients) ten periodontal defects received cultured gingival fibroblasts (GF) on the β-TCP scaffold and covered by a collagen membrane. The clinical evaluation was carried out at the beginning and at 6 months. Gingival crevicular fluid (GCF) samples were collected directly from the test sites for the quantitative measurement of PDGF-BB and BMP-2 using the ELISA kit at 1, 7, 14, and 21 days after surgery. Results Group II reported a significantly greater reduction in vertical pocket depth (VPD) and CAL gain compared with group I after 6 months. Radiographic bone gain was statistically higher in group II compared with group I. A significantly higher concentration of PDGF-BB was observed in group II on days 1, 3, and 7 compared with group I. Conclusions Translocation of gingival fibroblasts from gingival tissue to periodontal defects could be a promising option that increases cellular elements with regeneration potential. The concept of total isolation of gingival fibroblasts using occlusive membranes must be re-evaluated.

Background and objective The aim of this interventional, parallel-assignment, single-blinded, randomized, efficacy study was to investigate the efficacy of the treatment of gingival recessions by osteogenic cell transfer. This is the first randomized clinical trial of this nature. Material and methods Treatment of Miller class I or II gingival recessions >4 mm was randomly defined by casual sorting and performed by newly forming bone graft (NFBG) and coronally advanced flap (CAF) or subepithelial connective tissue graft (SCTG) and CAF (control). Clinical examinations were performed by a single blinded examiner at baseline and at 3, 6, and 9 months after surgery. Parameters investigated were recession height (REC), probing depth (PD), clinical attachment level, gingival bleeding index (GBI), plaque index (PlI), and keratinized gingiva width (KGW). Results Both techniques achieved similar percentage of root coverage, but NFBG was more effective in pocket depth reduction, attachment level gain, and reduction of plaque accumulation and of bleeding on probing. Conclusions These findings suggest that the transfer of osteogenic cells by NFBG is able to improve clinical attachment level and to cover denuded roots. NFBG can be used as an alternative treatment of deep recessions (≥4 mm), where the restoration of lost periodontal tissues is intended.

Background and objective The periodontal ligament (PDL) is an essential tissue for tooth function. However, the 3-dimensional ultrastructure of these PDL collagen bundles on a mesoscale is not clear. We investigated the 3-dimensional ultrastructure of these collagen bundles and quantitatively analyzed their histomorphometry using focused ion beam/scanning electron microscope (FIB/SEM) tomography. Material and methods The PDLs of the first mandibular molar of male C57BL/6 mice were analyzed using FIB/SEM tomography. The serial images of the collagen bundles so obtained were reconstructed. The collagen bundles were analyzed quantitatively using 3-dimensional histomorphometry. Results Collagen bundles of the PDL demonstrated multiple branched structures, rather than a single rope-like structure, and were wrapped in cytoplasm sheets. The structure of the horizontal fiber of the collagen bundle was an extensive meshwork. In contrast, the oblique and apical fibers of the collagen bundle showed a chain-like structure. The area and the minor and major axis lengths of cross-sections of the horizontal fiber, as determined from 3-dimensional images, were significantly different from those of the oblique and apical fibers. Conclusion These findings indicate that collagen bundles in horizontal fiber areas have high strength and that the tooth is firmly anchored to the alveolar bone by the horizontal fibers, but is not secured evenly to the alveolar bone. The tooth is firmly anchored around the cervical area, creating a "slingshot-like structure." This study has provided further insights into the structure of the PDL and forms the basis for the development of more effective therapies for periodontal tissue regeneration.

Background and objective Periodontal disease is a chronic inflammatory disease caused by bacterial infection that can lead to tooth loss. Gingival crevicular fluid can be collected easily and noninvasively. We previously discovered the presence of apolipoprotein B (apoB), the main constituent of low-density lipoprotein, and oxidized low-density lipoprotein (oxLDL) in the gingival crevicular fluid of healthy subjects. In this study, we investigated whether periodontal conditions affect the levels of apoB and oxLDL in gingival crevicular fluid. Material and methods The study population comprised 11 patients with chronic periodontitis. A pair of gingival crevicular fluid samples was collected from each patient at a healthy site and at a site with periodontitis (baseline samples). Thereafter, gingival crevicular fluid samples were collected from the same patients again at 4 and 8 wk after scaling and root planing (SRP). The levels of apoB, oxLDL, protein and cytokines in gingival crevicular fluid, in addition to gingival crevicular fluid volume, were measured. Results At baseline, the levels of apoB and oxLDL in gingival crevicular fluid were higher at the sites with periodontitis than at the healthy sites. The levels of apoB and oxLDL at periodontal sites decreased after SRP. The level of oxLDL in gingival crevicular fluid correlated well with the probing pocket depth. The oxLDL : apoB ratio in gingival crevicular fluid was significantly higher than that in plasma. Conclusions The levels of apoB and oxLDL in gingival crevicular fluid change according to the periodontal tissue conditions.

Stavropoulos A, Wikesjo UME. Growth and differentiation factors for periodontal regeneration: a review on factors with clinical testing. J Periodont Res 2012; 47: 545–553. © 2012 John Wiley & Sons A/S Background and Objective: A large body of evidence implies that growth and differentiation factors, based on their ability to regulate various functions of cells originating in the periodontal tissues, may support periodontal wound healing/regeneration, creating an environment conducive to and/or immediately inducing de novo tissue formation. This study presents a short systematic overview on growth and differentiation factor technologies evaluated in the clinic for their potential to enhance periodontal wound healing/regeneration. Material and Methods: Reports on growth and differentiation factor technologies evaluated in the clinic for their potential to enhance periodontal wound healing/regeneration were selected for review. Results: Growth and differentiation factor technologies intended for periodontal wound healing/regeneration and evaluated clinically included platelet-derived growth factor, insulin-like growth factor-I and -II, basic fibroblast growth factor, bone morphogenetic protein-3 and growth differentiation factor-5; platelet-derived growth factor was the only Food and Drug Administration-approved commercially available growth and differentiation factor technology. In general, enhanced periodontal regeneration was observed in sites receiving growth and differentiation factors compared with control(s). However, improvements of relatively limited clinical magnitude have been shown thus far. Conclusion: Although growth and differentiation factors project considerable appeal as candidate technologies in support of periodontal wound healing/regeneration, current candidate and commercially available technologies enhance treatment outcomes only to a limited extent in clinical settings.

The three-dimensional physiological tooth movement synchronized with heartbeat, periodontal pulsation, has already been demonstrated by previous researchers. The purpose of this study was to estimate physiological tooth movements within clinically normal periodontal tissues, thus establishing periodontal pulsation standards. The measuring device consisted of an amorphous sensor and a small magnet, which could detect tooth displacement without coming into contact with the tooth surface. The samples consisted of the left maxillary central incisors of 23 adult subjects. Three points on the labial surface were used as reference points to measure the labial-lingual movement range, and a point on the incisal edge was used for measuring the incisal-apical movement range. Periodontal pulsation and electrocardiogram measurements were recorded simultaneously for six minutes. The results showed that the mean amplitude of periodontal pulsation concerning the labial-lingual movement range was equal at the three labial points (0.59 microm) and tended to be smaller than that of the incisal-apical movement range (0.87 microm). There were no significant differences in the mean amplitude of periodontal pulsation at any of the four reference points. Significant correlations of the amplitude of periodontal pulsation existed among the three labial points. Though most teeth pulsated uniformly to the labial direction, some teeth moved in a complex manner including rotation with an axis among the three labial points. These results suggest that the values of periodontal pulsation amplitude which were recorded employing the amorphous sensor device are reliable standards of periodontal pulsation within clinically normal periodontal tissues.

Lymphatic drainage and circulation in periodontal tissues have been cited as important components of host defence and pathogenic mechanisms, but quantitative data are sparse because of the technical difficulties associated with small animal lymphatic studies. However, the lymphatic vessels draining the periodontal tissues and surrounding region are sufficiently large in sheep to permit surgical placement of lymphatic catheters. Consequently, lymph and recirculating lymphocytes can be continuously collected and this permits the quantitative assessment of local immune responses in these tissues. We have studied the lymphatic drainage pathways from the labial gingival tissues in sheep by two methods. First, in a series of anatomical studies (n = 6), a complex of Evan's blue dye and albumin was injected into the labial gingival tissues. One hour after injection the animals were sacrificed and the submandibular and cervical regions were dissected to expose the stained lymphatics. This anatomical study demonstrated 2 major drainage pathways: 1) cervical lymph ducts and; 2) efferent prescapular lymphatics. Secondly, to compare the relative importance of these two drainage pathways, radiolabeled protein (125I-albumin) was injected directly into the gingival tissues and its appearance in the cervical and prescapular lymph was measured (n = 7). Despite the technical difficulties encountered in the experiments, data collected showed that over 7.5 h, 64.7% of the injected protein was recovered in the prescapular and cervical lymph vessels (31.8 +/- 6.5% and 32.9 +/- 8.5%, respectively). In addition, 11.9 +/- 2.1% of the injected protein was transported to the blood by routes not involving the cannulated cervical and prescapular lymph vessels. With most of the remaining radiolabeled protein (17.9 +/- 4.9%) recovered from the injection site, we were able to account for approximately 95% of the injected protein. This study suggests that the lymph drainage from this region in the sheep model could provide one of the best described closed and contained systems and thus, could be a useful system for future continuous monitoring of inflammatory responses during experimental periodontal diseases.

Background and Objective Periodontitis is more frequently found in subjects with Down's syndrome. The aim was to investigate whether the relationship between MMPs and TIMPs) in the gingival crevicular fluid of subjects with Down's syndrome is altered compared with controls. Material and Methods Twenty-one adolescents with Down's syndrome and gingivitis (DS-G), 12 subjects with Down's syndrome and periodontitis (DS-P), 26 controls with gingivitis (HC-G) and eight controls with periodontitis (HC-P) were clinically examined. All patients were between 11 and 20 years of age. Gingival crevicular fluid was collected from each subject and the concentrations of MMPs (2, 3, 8, 9 and 13) and TIMPs (1, 2 and 3) (expressed as pg/μL adjusted for volume1 of gingival crevicular fluid) were determined using multianalyte kits from R&D Systems. Results The concentrations of MMP-2, MMP-3, MMP-8, MMP-9 and TIMP-2 in gingival crevicular fluid were significantly higher (p

Background and Objective Electric current is used to promote wound healing. However, it is unclear whether electrical stimulation contributes to gingival tissue remodeling. This study examined the effects of electrical stimulation on gingival tissue remodeling in a rat periodontitis model. Material and Methods Male Wistar rats (n = 28, 8 wks of age) were divided into four groups of seven rats each. The control group did not receive any treatment for 6 wks. In the other groups, periodontitis was ligature-induced for 4 wks. After 4 wks, the rats with periodontitis were given daily electrical stimulation of 0, 50 or 100 μA for 2 wks. Results The periodontitis group stimulated with 0 μA showed a higher density of polymorphonuclear leukocytes and a lower density of collagen in gingival tissue compared with the control group (p

The purpose of the present study was to study histologically and histometrically the inhibitory effect of aspirin on root resorption induced by mechanical injury of the periodontal soft tissues in rats. Resorption lacuna in the root surface of the molar in animals given both mechanical injury and aspirin administration contained fewer odontoclasts and was smaller in length and area than that in animals given only mechanical injury. The result of the present investigation indicated that the administration of aspirin might suppress root resorption induced by mechanical injury of the periodontal soft tissues.

Data from several sources demonstrate that disease-active and disease-inactive periodontal pockets exist, but currently available diagnostic procedures do not permit identification of disease-active sites at any given point in time. Using the experimental gingivitis model, we have performed studies aimed at determining whether levels of the enzyme aspartate aminotransferase (AST) in gingival crevicular fluid correlate with the presence and extent of periodontal inflammation. Gingival inflammation was assessed using the Gingival Index and the Sulcular Bleeding Index, and enzyme activity was measured using a standard procedure. Our data reveal a statistically significant association between AST values and Gingival Index scores for spontaneously occurring lesions (p less than 0.02-0.04) and experimentally induced lesions (p less than 0.0001), as well as the extent of change in these values during developing experimental gingivitis (p less than 0.0001) and resolving experimental gingivitis (p less than 0.0001). The data demonstrate that AST levels can be used to assess the presence and extent of periodontal inflammation.

The use of synchrotron X-ray diffraction has enabled us to record for the first time the first few orders of the collagen meridional low angle pattern from small, weakly diffracting native specimens of human periodontal ligament and gingival connective tissue, and to measure the D-period (fibrillar axis molecular overlap plus hole repeat) accurately. Comparison with human and other mammalian tendon and dermal specimens indicates that the D-period in both periodontal tissues resembles the value for dermal collagen, 65 nm, rather than that for tendon collagen, 67 nm.

Established chronic gingival lessions were studies in four categories of patients. One groups of healthy subjects was kept plaque-free (CO), while in the second group (CP) moderate accumulations of dental plaque were permitted. The third group (UH) comprised patients with uremia and in hemodialysis. A fourth group (IS) had received renal allografts and was kept on an immunosuppressive regimen. Differential blood counts and serum immunoglobin quantitation from the UH and IS groups gave mean values within normal ranges. Gingival biopsies were obtained from each subject. The connective tissue inflammation (CTI) scores were compared between the groups. Despite abundant local plaque accumulations, the UH group displayed essentially the same CTI scroes as plaque-free controls (CO), while the IS group showed a significantly lower (p < 0.05) CTI score than the two in question. The CTI scores of the CP group (controls with plaque) were significantly higher (P < 0.01) than those of the UH, IS and CO groups. It is concluded that immunologic homeostasis is important in the periodontal tissue of man.

In the present study, the effect of bacterial protease on the periodontal tissues in rats was investigated histologically and histometrically. In the control group, 0.03 ml distilled water was applied to the lingual marginal gingiva of the mandibular incisor while, in the experimental group, 0.03 ml bacterial protease (distilled water with 300 units, Sigma type XIV) was applied to the same area. Both groups were treated once a day for 1, 3, 6, 9, 15 and 21 d. Histologically, the junctional epithelium in the experimental group showed a marked apical proliferation along the tooth surface, and the gingival connective tissue displayed also a slight inflammatory cell infiltration subepithelially. The remaining periodontal tissues were almost similar to those of the control group. Histometrically, the length of the junctional epithelium between the base of the gingival crevice and the most apical portion of the junctional epithelium in the experimental group at 9, 15 and 21 d were significantly greater (p less than 0.01) than those of the control group, respectively.

A method, combining the standardization of radiographs with consistent radiopaque marker placement using a controlled force, is presented. Ten adult volunteers with healthy mouths were selected. Customized radiographic film holders were fabricated consisting of a plastic stent of the maxillary or mandibular arches to which a radiographic bite-block was attached. An orthodontic stainless steel tube was added and placed so that the radiographic marker could be guided repeatedly to the base of the sulcus in the same position. The radiographs were exposed using a self-adhesive grid on the periapical film. To document repeatability, measurements using the device were obtained at different time intervals: twice-a-week for the first, third, and fifth week for a total of six measurements for each volunteer. Results showed that the mean vertical and horizontal dimensions were nearly constant across the study period. The device had been shown to be accurate and enabled placement of a radiographic marker to repeatedly reach the same position in the sulcus with a good degree of precision.

Pool expansion - a method for measuring in vivo collagen production that involves injection of excess unlabeled proline along with [3H]proline - was used to calculate the values for collagen half-life in rat: incisor and molar periodontal ligament, molar gingiva and palatal mucosa. Collagen in most periodontal tissues (except for incisor gingiva) turns over more rapidly than skin collagen. The results reaffirm the concept that collagen in most periodontal tissues turns over with unusual rapidity.

193 schoolchildren 15 years of age were grouped as non-smokers and smokers on the basis of their smoking habits. In all cases an X-ray examination was made of the lower incisors, and a record was made of plaque and gingival index. The resulting values were compared between non-smokers and smokers. The following conclusions could be drawn. Oral hygiene and gingival inflammation are directly correlated. Oral hygiene is worse in smokers than in non-smokers. The gingival status is worse in smokers than in non-smokers. When investigating gingival status on the same level of oral hygiene, no significant difference is found between the groups.

The effect of feeding normal rat cake, high sugar fat supplemented and high sugar fat-free diets on the periodontal tissues of rats has been studied. It was found that feeding a fat-free diet resulted in periodontal changes. The effect is considered to he due to the systemic effect or essential fatty acid deficiency with an associated contribution from impaction in the gingival crevice due to the physical consistency of the fat-free diet.

Collagen biosynthesis, as measured by conversion of 3-4 3H-proline to 3H-hydroxyproline, was studied in guinea pig oral tissue and skin during normal growth, ascorbic acid depletion and following administration of ascorbic acid to scorbutic animals. Collagen breakdown in oral and skin tissue of normal animals was determined by measuring the depletion of 14C-hydroxyproline over a seven month period of time. Oral collagen demonstrated a high level of metabolic activity, thus providing biochemical support to the histo-pathological concept that periodontal tissues exist in a state of continuous wound healing and repair.

Three experimental studies were carried out to compare the effect of different implements recommended for interdental cleaning. In young adults, with healthy periodontal tissues, dental floss was superior to toothpicks in removing plaque from the lingual parts of the interproximal surfaces. The use of a single-tufted brush as a supplement to the toothpicks compensated for the lack of effectiveness of toothpicks alone. In wide-open interproximal areas, following periodontal destruction, the interdental brush is the most suitable implement to remove bacterial plaque.

Serum from dental patients was examined for its capacity to affect migration of polymorphonuclear leukocytes toward a complement-independent chemotactic factor, bacterial filtrate of Escherichia coli, (BF), Seven subjects with advanced periodontitis, documented with clinical measurements of pocket depth, loss of attachment, indices for gingival inflammation and oral hygiene, were paired with normal subjects of similar age and sex. This study shows that patients whose only apparent clinical symptom was severe periodontal inflammation harbor a heat-stable, serum component which neutralizes factors chemotactic for polymorphonuclear leukocytes. A role for this component in modulation of inflammatory infiltration is discussed.

The present investigation was carried out in order to study some aspects of the clinical, roentgenographical and histopathological alterations of periodontal tissues in dogs which during a 4-year period were allowed freely to accumulate plaque. Twenty inbred Beagle dogs, at the start of the study 10 months of age, were used. During a preparatory period of eight weeks the animals were once weekly subjected to a careful prophylaxis and had their teeth brushed twice daily. At the end of this period the dogs were divided into two groups of ten (test and control). From day zero of experimentation and onwards the teeth of the control dogs were twice daily subjected to meticulous toothbrushing, whereas the teeth of the test dogs were not cleaned. Examinations of the periodontal tissues were performed at days 0, 7, 14, 21, 28 and after 2, 4, 6, 8, 12, 18, 24, 36 and 48 months. Biopsies of different tooth regions were made on day zero and after 6, 12, 18, 24, 36 and 48 months of experiment. In the sections were measured (i) the distance from the cemento-enamel junction (CEJ) to the most apical cells of the dento-gingival epithelium, and (ii) the distance from CEJ to the level of the marginal alveolar bone. The study demonstrated that it is possible in dogs to establish and maintain a normal gingiva simply by eliminating calculus and then subjecting the animals to daily repeated and carefully performed tooth cleanings. Dogs allowed freely to accumulate plaque rapidly developed signs of gingivitis and eventually also clinical, radiographical and histopathological signs of periodontal tissue breakdown. The observations show that at least one type of periodontal disease is induced by factors within the dental plaque.

In an investigation of the effect of prolonged daily supervision of toothbrushing on Swedish schoolchildren (Koch and Lindhe, 1965; Lindhe and Koch, 1966) the incidence and severity of gingivitis in the experimental group were found to be substantially less than in the control group. It was therefore thought of interest to ascertain if there were roentgenographically detectable differences between the groups. The present investigation was carried out on 64 Swedish scoolchildren, randomly picked from the larger material taking part in the above mentioned investigation. Of these, 33 belonged to the supervised group and 31 to the group serving as a control. The criteria used for the diagnosis of periodontal disease in the roentgenograms were: 1 marginal tickening of the periodontal membrane. 2 defects of the margin of the alveolar crest. 3 increased distance between the cemento-enamel junction and the peak of the alveolar crest. Roentgenographs of the upper and lower front teeth were obtained using a modified parallel technique. The roentgenographic interpretation was carried out as a blind test. No statistically significent differences between the two groups were found. Because of these findings, roentgenograms of the 14 cases who showed no signs of gingival inflammation and of the 10 cases who had the highest gingival scores were selected. These roentgenograms were re-examined simultaneously by two of the authors. As before, no statistically significant differences were found between the two groups.

Fifty-six, female, BNL strain mice, 26 weeks of age, received a modified “gingiveclomy” excising, the gingival papilla mesial to the maxillary left first molar. The right maxillae served as the uninjured control site. Additional non-injured mice were utilized as further controls. At varying time intervals, groups containing 4 mice each were sacrificed over a 3 months experimental period. One hour prior to sacrifice, each animal received a subcutaneous injection of 1 uCi of H3TDR/gm of body weight. At sacrifice, both left and right maxillae were dissectedd out and prepared for histologic and autoradiographic study. Following injury, the highest proliferative activity was encountered at the epithelial wound edge. This activity became prominent about 8 to 15 days after injury, while the adjacent crestal epithelium demonstrated a markedly increased activity as early as 16 hours after wounding. The reformed junctional and crevicular epithelium, and adjacent fibroblasts, still exhibited elevated activity at one month post-injury and returned to control range values between one and three month following wounding. The remaining fibroblasts, osteoblasts and cementoblasts around the wound periphery showed markedly elevated activity as early as one to two days post-injury which returned to control levels between 2 to 4 weeks after wounding.

The changes in the periodontal tissues of the rat, when submitted to inanition, were studied at body weight losses of 4, 8, 12, 20, 24, 28, 32, 36, and 40 per cent. Histological and histochemical methods were used to investigate the effects. It was possible to establish that the tissue changes started in the middle portion of the periodontal membrane, migrating in the beginning towards the alveolar bone and only in a later stage towards the cementum. There were no structural changes in the gingival mucosa. The results obtained are discussed in the light of the varying known metabolic rates of the various zones.

To determine any effect the use of an oral contraceptive may have upon the periodontal tissue of rats Envoid E (25 parts norethynodrel and 1 part mestranol) was administered in daily doses of 104 mcg or 416 mcg/100 g body weight subcutaneously (except Saturday and Sunday) to 61 day old rats for 91 days. At the end of the 91 days the rats were killed and examined. Bacteriological analysis of the teeth and oral cavity revealed 106 colonies none of which were judged detrimental to periodontal tissue. Absolute amounts of bone loss were determined in the mandibular and maxillary bone. Maxillary bone loss was not significantly different among the high or low dosage groups or the control group which did not receive doses of the drug. However the mandibular bone loss was significantly less in the high dosage group than in the control group (105.2 plus or minus S.E. vs 120.2 plus or minus S.E.). The combined mandibular and maxillary bone loss of the rats receiving the 104/mcg dose was significantly less than that of the control group. Histological examinations revealed little difference between the controls and the 2 treated groups. The difference between the mandibular bone loss (significant) and the maxillary bone loss (insignificant) in the groups receiving Envoid E doses cannot be explained. Both high and low dosage rats experienced reduction in growth hormones caused by the steroids. Otherwise the use of Envoid E did not have a detrimental effect upon the periodontal tissue.