Comparative evaluation of decalcified freeze dried bone allograft with and without local doxycycline in non-contained human periodontal infrabony defects.

BACKGROUND: Doxycycline has been advocated as useful adjuncts in periodontal therapy not only due to their antimicrobial actions, but also to their recently recognized anti-collagenolytic, anti-inflammatory, osteoclast inhibitory and fibroblast stimulating property. The purpose of the present cohort study was to evaluate the regenerative outcomes of bone graft with or without local doxycycline in non-contained infrabony periodontal defects.
MATERIALS AND METHODS: 16 one or two wall infrabony defects, in 11 patients suffering from moderate to severe chronic periodontitis, aged 35-60 years, were randomly divided for bone graft, alone (control) and with doxycycline (test) for the study. At baseline, after 3 months and after 6 months of post-operative period, pocket probing depth (PPD), clinical attachment level (CAL), radiological bone fill (RBF) and alveolar height reduction were recorded. Analysis of variance and Newman-Keuls post-hoc test were used or statistical analysis. A two-tailed probability (P) value P < 0.05 was considered to be statistically significant.
RESULTS: For the control group PPD reduction 2.00 ± 0.18 mm, CAL gain 1.38 ± 0.17 mm, RBF 0.63 ± 0.27 mm (18.0%) was observed while in the test group PPD reduction 2.00 ± 0.38 mm, CAL gain 1.25 ± 0.31 mm, RBF 0.75 ± 0.31 mm (20.7%) was evaluated. While alveolar height reduction for the control group and test group was 13% and 12.5% respectively.
CONCLUSION: The study confirmed no added benefits of local doxycycline, as compared with bone graft alone, for regeneration of non-contained human periodontal infrabony defects.

INTRODUCTION

Treatment of infrabony defects with different grafting materials has provided a reliable method of achieving defect fill. Recent studies indicate, however that regrowth of osseous tissue alone does not necessarily lead to periodontal regeneration.[12] In fact, the most common healing pattern following the graft procedure is by long junctional epithelium.[12] New attachment and regeneration of the periodontium may be facilitated when the healing area is selectively repopulated with the periodontal ligament cells and methods of obtaining selective cell repopulation is by use of an occlusive membrane and by chemical means.[34]

Tetracyclines constitute a family of antibiotic that have been found to be effective against the putative periodontopathogens and has been shown to initiate demineralization on the bone surface layer, which results in the release of osteogenic factors such as the transforming growth factor, insulin-like growth factor or the bone morphogenic proteins that trigger bone induction.[5] Doxycycline, a member of the tetracycline family, has the capability of conditioning dentine and also enables fibrin linkage, both of which favor the formation of a new attachment.[67] Doxycycline potentiates osseous regeneration in periodontal defects when locally administered due to its anti-collagenolytic and antiproteolytic properties, which enhances the bone forming ability through osteoblast cell chemotaxis and reduced bone resorption.[8] Tetracycline, in combination with bone grafting procedure, has been successfully used for regeneration of periodontal infrabony defects.[910]

The current widespread use of decalcified freeze dried bone allograft (DFDBA) is based on the purported osteoinductive ability of bone graft preparations. Demineralization of the graft exposes the bone inductive proteins located in the bone matrix and in fact, may activate them.[11]

There is no consensus in previous studies about the use of antibiotic with bone grafts in infrabony periodontal defects.[9101213]

In the literature, no study is present regarding the benefits of local doxycycline, in terms of regenerative potential, with bone graft in unfavorable infrabony periodontal defects. Hence, the purpose of the present study was to compare the regenerative outcomes of DFDBA use alone and in combination with local doxycycline in non-contained human periodontal infrabony defects.

MATERIALS AND METHODS

16 one or two wall infrabony periodontal defect in 11 patients (5 males and 6 females) aged between 35 and 60 years suffering from moderate to severe chronic periodontitis, were selected for the study at the Department of Periodontics, Institute of Dental Sciences, Bareilly. The inclusion criteria for the patients were absences of any systemic diseases, not taking any medication, no pregnancy or lactation, non-smokers, previously not treated for periodontal reasons, absence of furcation involvement, presence of at least one infrabony defect with 1/2 wall infrabony component ≥ 3 mm and pocket probing depth (PPD) ≥5 mm, (as assessed through bone sounding). Only multi-rooted teeth involved in the present study. Altogether, two maxillary first molars, two maxillary second molars and four mandibular first molars represented the control sites (DFDBA alone); three maxillary first molars, two maxillary second molars and three mandibular first molars were selected as the test group (DFDBA with doxycycline). For the combined graft, doxycycline pellets and DFDBA were mixed using a 1:4 ratio (doxycycline: DFDBA) by volume and then laced in the defect. Patients were explained about the study, risks, benefits and procedures and a written informed consent was obtained from every patient. All the examinations, treatments and procedures associated with this study followed the principles according to the Declaration of Helsinki. The study was properly reviewed and approved by the Ethical Committee of Institute of Dental Sciences, Bareilly. All patients underwent initial therapy consisting of full mouth scaling and root planning, oral hygiene instructions and occlusal adjustment when indicated. 4 weeks following completion of initial therapy, a re-evaluation examination including the evaluation of the percentage of gingival sites exhibiting the presence of bacterial plaque (%PL-), bleeding on probing (%BOP+), PDD and clinical attachment level (CAL) was carried out in each patient. Patients were scheduled for surgery after demonstrating lesser than 20% PL+ and BOP+ sites. The base line parameters associated with the defects had no significant difference between control and test group. These defects were analyzed clinically and radiologically at baseline, 3 months and 6 months following regenerative surgery by a single investigator for each surgical site.

Clinical and radiological parameters

PPD and CAL were recorded to the nearest millimeter with a standardized periodontal probe (UNC-15 probe) at the deepest point of the periodontal pocket on each tooth to be treated from the vertical groove cut in the acrylic stent made on the occlusal surface.

Radiographical bone fill and resorption of alveolar bone height were measured as the distance from a fixed reference point, i.e. radiographic cemento-enamel junction. Radiographic examination was carried out by intraoral periapical radiographs, which were taken using the parallel cone technique with a customized film holder. An effort was made for both pre-operative and post-operative radiographs to have a close projection geometry and similar optical density. Parameters were recorded using a radiological grid (1 mm × 1 mm) in the nearest millimeter [Figures 1–4].

Figure 1

Base line radiograph for the test group

Figure 4

Post-operative radiograph for the control group

After 6 months radiographs for the test group

Pre-operative radiograph for the control group

Surgical procedure

The standard surgical procedure consisted of intrasulcular incision, full thickness mucoperiosteal flap reflection, thorough defect debridement and root planning with both ultrasonic and hand instruments. DFDBA (LifeNet Health, Virginia Beach, VA, USA) was reconstituted with sterile saline water and placed into the defect up to the alveolar crest. For the combined graft, doxycycline pellets and DFDBA were mixed using a 1:4 ratio (doxycycline: DFDBA) by volume then placed in the defect. For flap closure, 3-0 black braided silk sutures were given [Figures 5–8]. All surgical sites were dressed with periodontal dressing for a period of 10 days, after that the dressing was removed and suture removal was accomplished.

Figure 5

Base line probing pocket depth for the test group

Figure 8

Suturing after flap repositioning

Infrabony defect after flap reflection

Bone graft with doxycycline placement in infrabony defect

Post-surgical care

The post-operative regimen included both verbal and written instructions. Patients were prescribed amoxicillin 500 mg and 125 clavulanic acids 8 hourly for 5 days and 0.12% chlorhexidine gluconate mouth rinse, twice a day for 2 weeks. All patients were placed on a strict maintenance schedule following surgery. At every 2 weeks appointment, total professional plaque removal was performed and personal oral hygiene was reinforced and practiced.

RESULTS

After 6 months of post-operative period, statistically significant clinical and radiological differences were revealed as compared with base line, in both groups. On 3 months to base line comparison statistically, significant improvement presented in soft-tissue parameters only. And for comparison of 6 months to 3 months significant change in hard tissue parameters was revealed. On intergroup comparison, there were no significant differences present for any parameter at any time period [Tables 1 and 2].

Table 1

PPD, CAL, RDD, ABH (mm) summary (mean±SE, n=8) and significance (P value) of mean difference between groups at three different periods

Table 2

For each group, significance (P value) of mean difference in PPD, CAL, RDD, ABH between the periods (within groups)

All patients maintained a high standard of oral hygiene care during the study and all the defects healed uneventfully. Both group showed no significant change in %PL + and %BOP + during baseline and 6 months period.

For the control group PPD reduction 2.00 ± 0.18 mm, CAL gain 1.38 ± 0.17 mm, radiological bone fill (RBF) 0.63 ± 0.27 mm (18.0%) was observed and for the test group PPD reduction 2.00 ± 0.38 mm, CAL gain 1.25 ± 0.31 mm, RBF 0.75 ± 0.31 mm (20.7%) was evaluated. While alveolar height reduction for the control group and test group was 13% and 12.5% respectively.

DISCUSSION

The results of this study suggested that the addition of doxycycline did not have any appreciable influence on any soft-or hard tissue clinical parameter. Bone graft combined with local doxycycline was associated with more bone fill than bone graft alone. However, this difference was non-significant at any time period. On intra group comparison, both group revealed significant improvements in parameters from base line. Previous studies with DFDBA in non-contained infrabony defects showed significant benefits as per our study[67] while some have found no benefit in the use of DFDBA.[14]

The findings of this study, follow the Masters et al.[9] They determined a significant improvement in clinical parameters of infrabony defects treated by DFDBA with tetracycline after 1 year. However, their study suggested that there is no significant benefit from reconstituting the allograft with 50 mg/ml of tetracycline hydrochloride as compared with DFDBA alone. Clinical studies that have demonstrated beneficial effects of tetracycline in periodontal bone grafting therapy are contradictory to this study.[101213]

A triple substantive benefit of tetracyclines may be derived due to antimicrobial, anti-collagenolytic and fibroblast stimulating activities.[515] The antimicrobial component makes tetracycline the drug of choice in periodontal therapy while the anti-collagenolytic effect may act to prevent root and bone resorption. Increased fibronectin binding to tetracycline conditioned-roots promotes the attachment of fibroblasts and inhibits epithelial cell attachment.[161718]

The bacteria that are usually associated with periodontal disease predominantly reside in a multispecies biofilm. Doxycycline has been found to be highly effective in inhibiting biofilm formation by various periodontopathogens.[14] Doxycycline has been found to inhibit collagenase and other host derived matrix metalloproteinase, which are released as the periodontal disease progresses.[15] It protects alpha-1 proteinase inhibitor from proteolytic inactivation in the gingival crevicular fluid and also inhibits the production as well as the scavenging of reactive oxygen radical generated by the polymorphonuclear neutrophils.[1920] In addition to this, doxycycline is a long acting tetracycline with less susceptibility to chelation than tetracycline hydrochloride.[21]

Adequate results have been reported on the association between the number of bony walls remaining (defect containing) and the regenerative potential of periodontal infrabony defects. We selected one or two wall periodontal infrabony defects for this study because various studies have proposed that the three wall infrabony defects inherently heal more favorably.[222324] A biological explanation of this healing pattern has been related to the fact that the number of bony walls of the defect will influence, among other factors, the source of osteoprogenitor cells. An alternative explanation, should be considered that one wall component, when present, is more exchangeable to surroundings, more susceptible to oral environmental and thus filling materials would have more chance to get contamination, factors leading to an incomplete bone fill.[24] Local doxycycline was added to bone graft in this study to overcome these shortcoming associated with bone graft in one wall infrabony defects.

The results of our small cohort study are less in magnitude from the result of the previous mentioned studies. Difference in the outcomes of the various studies might be due to the variation in several associated factors that can alter the extent of clinical attachment gain and bone regrowth following a grafting procedure, such as patients selection, defects characteristics, data collection, biochemical characteristics of grafted materials, surgical variation and patient attitude toward the treatment.[2324]

There is a significant variation in the inherited osteogenic potential of DFDBA.[2526] The regenerative potential of DFDBA therapy governs according to the osteoinductivity of the bone graft samples. These differences may be an according to age of the donor, previous exposure to pathology and/or drug therapy, genetic variation, length of time required to harvest the cadaver bone.[2627]

This study would have had more validity if defect width was also a consideration criterion. However, it would have been very difficult to provide similar defects with standardized depths, widths and morphology. A limitation of the present study was the number of defects treated; it would have been preferable to have a larger number of samples for study demonstrating statistically significant differences between the grafting groups with the same clinical trends. Further long term clinical trials, with larger samples need to be carried out to for documentation of regeneration of the lost periodontium.

CONCLUSION

In this small cohort study, addition of a local doxycycline to DFDBA did not have any additional benefits as compare with DFDBA alone for periodontal regeneration in non-contained periodontal infrabony defects.