Evaluation of autologous periodontal stem cells niche in the treatment of periodontal intrabony defects: A split-mouth randomized controlled trial

Abstract

Background:

Regeneration of the lost periodontium requires an abundant supply of the desired cell. Stem cells can provide progenitor cells at the target location; however stem cell therapy is technique sensitive. Thus, using periodontal ligament stem cells (PDLSCs) of an extracted tooth can act as a feasible and accessible alternative to an otherwise expensive elaborate process. Thus, the aim of this study was to evaluate the effects of direct transplantation of autologous PDLSC niche in two-walled or three-walled intrabony defects.

Materials and Methods:

A total of 16 patients having bilateral intrabony defects (total 32 sites) were recruited for the study. The socioeconomic status was also taken into consideration. After randomization, the sites were treated with either open flap debridement alone (control group) or open flap debridement followed by direct transplantation of autologous periodontal ligament stem cells niche (APDLSc Ni-test group). For each surgical site, clinical parameters were evaluated at baseline, 3, 6, and 9 months. Radiographic assessment was made at baseline, 6, and 9 months.

Results:

Both the groups have shown improvement from baseline to 9 months in clinical and radiological parameters, which was statistically significant. The intergroup comparison has shown a nonsignificant difference in favor of the APDLSc Ni group. However, bone fill was better with A PDLSc Ni than open flap debridement alone.

Conclusion:

The result of the present study concluded that treatment of intrabony defect by direct transplantation of autologous periodontal ligament stem cells niche was beneficial and can be used in a simple clinical setup.

INTRODUCTION

The regeneration of lost periodontium has always been a matter of interest; as a periodontist, the aim is to ensure the development of new bone, cementum as well as the periodontal ligament; however, it is a daunting task at hand. Melcher, in 1976, gave an interesting concept where he pointed out that in the case of periodontal defects, there is the presence of progenitor cells of all four tissues, which constitutes periodontium. The proposition exclaimed that regeneration could be achieved if the periodontal ligament cells populate the injured area before the cells of epithelium, connective tissue, and alveolar bone.[1] This produced an understanding that if unwanted cells are to be excluded, the possibility of regeneration increases. Numerous biomaterials and their combinations have been tried and tested;[2] the results are also satisfactory; however, the search for the best goes on. Based on what Melcher has to offer, the exclusion of cells is one way to approach the problem. Recent developments in stem cell therapy has shown that direct application of stem cell has shown promising results and therefore can serve as better optio.

The regenerative therapy aimed at using stem cells has been a matter of research in recent times. In this bequest, stem cells to regenerate dental tissues have been isolated and studied. Periodontal ligament stem cells (PDLSCs) were first obtained by Seo et al.[3] The cells have shown a close resemblance in their regenerative potential to that of mesenchymal stem cells. These cells are able to differentiate into chondrocytes, osetoblasts, and cementoblasts. They have also been shown to develop new periodontal ligaments when transplanted in the defect site.[4] Due to their immunomodulatory properties, such as osteodifferentiation (causing bone repair) and ability to produce fibrillar components similar to sharpeys fibers, these cells are studied extensively for neoformation of PDL.[5,6] The previous ex vivo and animal studies have shown that the use of PDLSCs in a suitable medium and vascular-rich environment is able to produce functional tissue regeneration in periodontally compromised areas.[7,8]

The isolation of PDLSCs is relatively easier than that of other dental tissue-derived stem cells. They can be isolated from third molars or any extracted tooth and have been shown to be directly implanted in the defect site using a carrier.[9] When cells from the periodontal ligament are transplanted in a defect site, it will create an environment where the signals from the extracellular matrix and growth factors of surrounding stimulate the PDLSCs to differentiate into desired cells to repopulate the area.[9] The technique used was “Stem cell Assistance In Periodontal Regeneration Technique (SAI-PRT)” where a direct application of root shavings having PDLSCs along with gel foam was done into the defect.[9] It is a relatively simplified and reliable method to induce the growth of stem cells in periodontal osseous defects compared to the extensive method of preparation of stem cell incorporated periodontal sheets.

The method of preparation is cheaper comparatively, so it can be well used in under-privileged population too. Thus, the present study was undertaken to assess the effectiveness of direct transplantation of Autologous Periodontal Ligament Stem Cell Niche (APDLScNi) in two- or three-walled intrabony defects, clinically and radiographically. The null hypothesis stated that both the modalities would have a similar effect on periodontal regeneration.

MATERIALS AND METHODS

A split-mouth single-blinded clinical study was approved by the institutional ethical committee. It was conducted in accordance with the Helsinki Declaration of 1975, as revised in 2013. Sample size calculation was performed based on results from Chen et al.[10] using an alpha (α) level of 0.05 and beta (β) level of 0.05, i.e., power = 95%. G power software, version 3.1.9.7 (Heinrich-Heine-Universitat Dusseldorf, Dusseldorf, Germany) was used for an A priori analysis of t-test. The effect size of 1.098 gave the sample size of 13 in each group; considering a 20% dropout, the final sample size was 16 sites per group. A written informed consent was obtained from all the patients, and the trial period was from November 2022 to March 2024.

Selection criteria

The patients selected had stage III Grade B periodontitis.[11] They were further curated based on inclusion criteria: age group of 25–50 years; systemically healthy patients; clinical attachment level ≥5 mm and probing pocket depth (PPD) ≥5–8 mm; vertical defects of ≥3 mm (two or three walled) assessed by transgingival probing and radiovisiography using long cone paralleling technique; no allergies to study materials; nonsmoker or smoking <5 cigarettes/day; no history of periodontal therapy in the past 6 months and having at least one tooth that needs to be extracted due to impaction or nonfunctional reasons which could be used for PDL stem cells. Patients excluded had unacceptable oral hygiene during the presurgical period, suffering from any known systemic diseases; pregnant or lactating females; patients unwilling for extraction; patients under medication known to affect periodontal tissues or healing.

Study design

The patients selected were those showing the presence of intrabony defects in at least two quadrants (16 patients, 32 sites). The randomization of sites was done using computer computer-generated method in the control and test groups. Once one of the sites was allocated to either of the group, the next chosen site automatically went to another group. The control group was treated with only open flap debridement (OFD) and the test group (A-PDLSc Ni) was treated with OFD followed by direct application of autologous PDLSC niche (A-PDLSC niche). The clinical parameters evaluated were: Plaque index (PI, Silness and Loe, 1964)[12] Gingival Bleeding Index (GBI, Ainamo and Bay, 1975)[12] PPD-measured from gingival margin to base of the pocket in millimeters using University of North Carolina (UNC) 15 probe with an acrylic stent used as a guide.[12] Relative attachment level (RAL)-measured from marking on the acrylic stent to the base of the pocket using UNC 15 probe [Figures 1a and 2a].[12] Gingival marginal position (GMP) – evaluated with an acrylic stent used as guide [Figures 1b and 2b][13] Keratinized gingival thickness (GT)– measured at midpoint location between gingival margin and mucogingival junction using endodontic reamer with stopper and then gauging it using digital Vernier calipers [Figures 1c, d, and 2c, d].[13] The clinical parameters were assessed at baseline, 3, 6, and 9 months.

Figure 1.

Test group. (a) Measurement of probing pocket depth at baseline, (b) Measurement of gingival marginal position, (c and d) Measurement of gingival thickness at baseline using Vernier caliper, (e) Intrasurgical measurement of probing pocket depth, (f) Harvested periodontal ligament (PDL) stem cells, (g) PDL Stems cells placed, (h) Sutures placed, (i) Measurement of probing pocket depth at 9 months, (j) Measurement of gingival marginal position at 9 months, (k and l) Measurement of gingival thickness at 9 months using Vernier caliper, (m) Preoperative RVG image at baseline, (n) Postoperative RVG image at 9 months

Radiographic parameters were assessed by radiovisiography and standardized using the Rinn positioning system. Linear measurements were taken as:[14]

• Cemento-enamel junction (CEJ) to bottom of the defect (CEJ to BD) = Defect Depth (DD)

• Bone crest change = (CEJ to AC [baseline])−(CEJ to AC [post op])

• Bone fill (BF) = (CEJ to BD [baseline])−(CEJ to BD [post op])

Socioeconomic status of the patient was also assessed using the Modified Kuppuswamy scale.[15] Radiographic parameters were assessed at 6 and 9 months to evaluate the bone DD, BF, and alveolar crest change.

Clinical procedure

All the patients underwent nonsurgical therapy to achieve appropriate gingival health. The surgical site was prepared using 0.2% povidone-iodine and anesthetized using 2% lignocaine containing adrenaline in a ratio of 1:80,000. The mucoperiosteal flap was reflected and debrided. Thorough removal of calculus and root planing was done by hand and ultrasonic instruments [Figures 1e and 2e]. In the case of the test group, stem cells were procured and grafted, then the flap was sutured [Figure 1f, g and h], while in the case of the control group, the flap was sutured back [Figure 2f]. Periodontal dressing was placed. Figures 1i-l depicts the recording of probing pocket depth, gingival margin level and gingival thickness at 9 months, post-surgery for test group. Figures 2g-j depicts the recording of probing pocket depth, gingival margin level and gingival thickness at 9 months post-surgery for control group. Figures 1m and n represents radiographs at baseline and 9 months, post-surgery for test group. Figures 2k and l represents radiographs at baseline and 9 months, post-surgery for control group.

Figure 2.

Control group. (a) Measurement of probing pocket depth at baseline, (b) Measurement of gingival marginal position at baseline, (c and d) Measurement of gingival thickness at baseline using Vernier caliper, (e) Intrasurgical measurement of probing pocket depth, (f) Sutures placed, (g) Measurement of probing pocket depth at 9 months, (h) Measurement of gingival marginal position at 9 months, (i and j) Measurement of gingival thickness at 9 months using Vernier caliper, (k) Preoperative RVG image at baseline, (l) Postoperative RVG image at 9 months

Method of preparation of A-PDLSc Ni: Vital tooth requiring extraction was obtained. The periodontal ligament tissue adherent to the tooth root and alveolar socket comprised PDLSCs along with its niche (PDL tissue niche). Components were gently removed using sterile curette and mixed with a gelatin sponge (Abgel^®©™) to form a transferable mass which was placed in the periodontal defect and was gently condensed [Figure 1g].

Routine postoperative instructions were given to the patients. 10 ml of 0.2% chlorhexidine digluconate solution twice daily was advised for mouth rinsing. Analgesics (ibuprofen 400 mg b. i. d. for 3–5 days) and prophylactic antibiotics (amoxicillin 500 mg t. i. d. for 5 days) were also prescribed. Periodontal dressings and sutures were removed 7–10 days after surgery. The patients were recalled for follow-up visits, and data were recorded. The tabulated data were subjected to statistical analysis using SPSS version 24 (the Statistical Package for Social Sciences, IBM) (SPSS Inc, Chicago, IL). Intergroup comparison at each interval was made using an unpaired t-test. The within-group comparison of BF at 6 and 9 months for both the test and control group was done using paired t-test. A P < 0.05 was considered statistically significant.

RESULTS

A total of 16 subjects, exhibiting two- or three-walled intrabony defects, of which seven subjects belong to the lower class, two subjects belong to the lower middle class and 7 subjects belong to the upper lower class. There were no adverse outcomes reported. The primary outcome measures were considered PPD, RAL, and GMP, and the secondary outcome measures were PI, GI, and GT. There was a significant improvement from baseline to 9 months in all the parameters for both the groups. PI and GI have shown significant reduction from baseline to follow-up intervals [Table 1]; there was no significant difference among the two groups [Table 2]; however, the test group showed a better reduction of PI and GI. Similar improvement in PPD and RAL was seen from baseline to 9 months in within-group comparison [Table 1 and Figures 1k 2i]; however, for both PPD and RAL, the intergroup difference at 3 and 6 months was significant, which became nonsignificant in favor of test group at the end of 9 months [Table 2]. The gingival margin position had shown improvement at 9 months in both groups [Table 1 and Figures 1l and 2j]. There was significantly better improvement in the test group than control at 9 months for GMP [Table 2]. GT was increased from baseline to 9 months for test and control groups both [Table 1 and Figures 1m, n, 2k, l]. There was a significant difference when intergroup comparison was done for GT [Table 1]. When DD and bone crest changes were evaluated from baseline to 9 months, there was a statistically significant improvement for both the groups, but the intergroup difference between the test and control was nonsignificant [Tables 1 and 2]. At 9 months, BF was significantly more in the test group than of the control group. However, within group comparison of BF has shown significant fill in the test group but nonsignificant fill in the control group [Tables 1 and 2, Figure 1o, p, 2m, n]. The results expressed the use of A-PDLSC Ni had a more favorable outcome as compared to OFD.

Table 1.

Intergroup comparison of all parameters done using unpaired t-test at various recall intervals

	Baseline, mean±SD	3 months, mean±SD	6 months, mean±SD	9 months, mean±SD
PI
Test	2.27±0.41	1.52±0.38	1.45±0.27	1.37±0.30
Control	2.28±0.40	1.67±0.34	1.45±0.30	1.45±0.30
P	0.939	0.282	0.506	1.000
GBI
Test	50.62±5.53	23.92±5.10	23.44±4.41	2.05±0.38
Control	50.91±5.61	26.51±5.24	24.27±4.51	2.05±0.38
P	0.895	0.214	0.639	1.000
PPD
Test	7.31±0.95	3.5±0.82	3±0.82	2.62±0.51
Control	7.31±1.18	4±0.82	3.46±0.52	3±0.82
P	0.999	0.005*	<0.001*	1.000
RAL
Test	8.62±0.96	4.15±0.80	4.08±0.87	3.62±0.51
Control	8.69±1.11	5±0.82	4.31±0.63	4.08±0.86
P	0.852	0.013*	0.005*	1.000
Gingival position
Test	6.69±1.03	6.18±0.95	6.00±0.90	5.68±0.77
Control	6.85±0.99	6.77±1.07	6.45±1.01	5.91±0.95
P	0.701	0.065	0.022*	0.012*
Gingival marginal thickness
Test	1.72±0.22	2.10±0.29	2.12±0.43	2.49±0.40
Control	1.70±0.25	1.92±0.22	1.95±0.35	2.31±0.40
P	0.841	0.091	0.302	0.252
DD
Test	9.01±1.05	-	4.84±0.54	4.35±0.64
Control	8.55±1.18	-	5.06±0.58	4.35±0.64
P	0.310	-	0.323	1.000
BC
Test	9.54±0.78	-	5.56±0.70	4.76±0.65
Control	9.69±1.11	-	5.00±1.01	4.00±1.00
P	0.686	-	0.110	0.477
BF
Test	-	-	4.17±0.83	4.58±0.90
Control	-	-	3.51±1.13	3.82±0.99
P	-	-	0.101	0.001*

*Significant difference amongst the groups (P<0.05). SD – Standard Deviation; P value – Probability value; BF – Bone fill; BC – Bone crest; DD – Defect depth; RAL – Relative attachment level; PPD – Probing pocket depth; GBI – Gingival bleeding index; PI – Plaque index

Table 2.

Intragroup comparison of all the parameters done using paired t test at various time intervals

Parameters	Follow up	Test, mean±SD	P	Control, mean±SD	P
PI	Baseline	2.27±0.41	<0.001*	2.28±0.40	<0.001*
	3 months	1.52±0.38		1.67±0.34
	6 months	1.45±0.27		1.45±0.30
	9 months	1.37±0.30		1.45±0.30
GBI (%)	Baseline	50.62±5.53	<0.001*	50.91±5.61	<0.001*
	3 months	23.92±5.10		26.51±5.24
	6 months	23.44±4.41		24.27±4.51
	9 months	2.05±0.38		2.05±0.38
PPD	Baseline	7.31±0.95	<0.001*	7.31±1.18	<0.001*
	3 months	3.50±0.82		4.00±0.82
	6 months	3.00±0.82		3.46±0.52
	9 months	2.62±0.51		3.00±0.82
RAL	Baseline	8.62±0.96	<0.001*	8.69±1.11	<0.001*
	3 months	4.15±0.80		5.00±0.82
	6 months	4.08±0.87		4.31±0.63
	9 months	3.62±0.51		4.08±0.86
GMP	Baseline	6.69±1.03	<0.001*	6.85±0.99	<0.001*
	3 months	6.18±0.95		6.77±1.07
	6 months	6.00±0.90		6.45±1.01
	9 months	5.68±0.77		5.91±0.95
GT	Baseline	1.72±0.22	<0.001*	1.70±0.25	<0.001*
	3 months	2.10±0.29		1.92±0.22
	6 months	2.12±0.43		1.95±0.35
	9 months	2.49±0.40		2.31±0.40
DD	Baseline	9.01±1.05	<0.001*	8.55±1.18	<0.001*
	6 months	4.84±0.54		5.06±0.58
	9 months	4.35±0.64		4.35±0.64
BC	Baseline	9.54±0.78	<0.001*	9.69±1.11	<0.001*
	6 months	5.56±0.70		5.00±1.01
	9 months	4.76±0.65		4.00±1.00
BF	6 months	4.17±0.83	<0.001*	3.51±1.13	<0.001*
	9 months	4.58±0.90		3.82±0.99

*Significant difference amongst the groups (P<0.05). SD – Standard Deviation; P value – Probability value; BF – Bone fill; DD – Defect depth; RAL – Relative attachment level; PPD – Probing pocket depth; GT – Gingival thickness; BC – Bone crest; GMP – Gingival marginal position; GBI – Gingival bleeding index; PI – Plaque index

DISCUSSION

Stem cell application, along with tissue engineering, has been proven pivotal in ensuring regeneration. The results of various in vivo and in vitro studies have shown success; however, the ex vivo process is highly technique-sensitive and expensive to apply, thus needing high-end equipment and infrastructure. If a better and cheaper alternative is available, it can become more feasible to use in patients along with underprivileged population. Vandana and Dalvi[16] have shown that when PDLSCs are implanted in the defect directly, are capable of producing regeneration, the present study also attempted to use the technique to achieve periodontal defect resolution.

Periodontal ligament stem cells have been cultured to show that they can proliferate into required multipotent cells for periodontal regeneration[3] and are termed SAI-PRT or stem cell assistance in the periodontal regeneration technique. They can be obtained from extracted tooth or socket lining. The root surface scrapings are also able to yield PDLSCs.[17] They have the ability to differentiate into fibroblasts, osteoblasts, and cementoblasts.[3] They have also been shown to reduce T-cells and obtain immunomodulation of healthy cells.[18] In the case of root scraping, there is the presence of cementum and dentinal chips, and this harbors a rich source of growth factors and bone morphogenetic proteins, which can accelerate PDL tissue formation. Dentinal calcified tissues mimic bone, leading to the differentiation of hard tissue and inhibiting inflammation.[16] When implanted on a carrier medium such as a gelatin sponge, this can serve as a scaffold and complete the triad of tissue engineering, providing an ideal recipe for assured regeneration.

The present study incorporated the root scrapings containing PDLSCs in a gelatin sponge implanted to periodontal osseous defects and compared with open flap debridement as control. The result has shown successful outcomes in terms of primary and secondary measures. The PI and GI were improved for both groups as oral hygiene measures and reinforcement was done. The difference for PPD and RAL was also achieved which may be the result of reduced inflammation due to debridement and removal of local factors in both groups. The defect fill had shown a significant increase in both the groups from baseline to 9 months, which was statistically significant in the test group as well as control group. The defect fill obtained could be accounted to the presence of insulin-like and fibroblast growth factors, which has been observed in PDLSCs in previous studies.[19]

The study was one of its kind, and previous literature does not justify the same methods and are hence not comparable. Although Feng et al.[20] and Chen et al.[10] had studied PDLSCs in periodontal defects but have used ex vivo culture and bone grafts to supplement regeneration. The study by Shalini and Vandana[9] used a similar setup; however, the results do not coincide with ours as defect fill has nonsignificant difference between groups, and improvement in density was significant for them. The reason to avoid ex vivo culture was due to the fact that processed cells may lose the ability to multiply; the process is extensive and expansive.[17] In order to achieve more reasonable treatment for economically backward patients, this autogenous implantation of stem cell therapy was used. PDLSCs, when transplanted in a matrix such as sponge does not only increase cell population but also get stimulated to proliferate specific tissues. The presence of integral signaling molecules directs the growth of cells required for regeneration. A similar speculation was proposed by authors,[9] who were successful in their attempt at regeneration, and the present study supports this as evidenced by the improvement of end parameters.

Although, radiographic evidence of defect fill was seen, histological confirmation of bone could not be done due to ethical concerns about the re-entry. The presence of the source of autologous PDL cells obtained from the extracted tooth is one of the limiting factors. The lack of quantification of cells that are reimplanted and would be active in the defect is yet another drawback that needs to be addressed. Further studies with larger sample sizes and the use of other matrices could be carried out to strengthen evidence.

CONCLUSION

The PDLSC have been the simplest source for progenitor cells as they are easy to extract as compared to others. They were reliable, as proven in many trials; thus, an attempt was made to further simplify the process in a clinical setup by direct application in the defect site. The use of autologous PDLSCs in periodontal defects has been proven effective in terms of improvement of clinical and radiographic parameters. This, when compared with open flap debridement, has been better due to the presence of innate cells and signaling molecules, which triggered appropriate pathways to achieve regeneration. The use of autologous implantation needs to be encouraged as not only it is feasible but more reachable to the general population.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.