Biological effect of the nanocrystalline calcium sulfate bone graft in the periodontal regeneration

Abstract

Background

The ultimate goal of tissue engineering is to reproduce functional alveolar∖Periodontal complex. This study aimed to asses micro- and nano-formulated crystalline calcium sulfate as possible treatment of intrabony defect utilizing an autologous Platelet rich fibrin scaffold.

Subjects

and Methods: An experimental prospective split mouth design using eight healthy mongrel dog’s was conducted. After two months of extracting the first premolar, bilateral intrabony defects were created mesial to the mandibular second premolar. One side was grafted by nanocrystalline calcium sulfate, while, the opposite side was grafted with microcrystalline calcium sulfate graft. Dogs were randomly euthanized after one month (group I) and 3 months (group II). The primary outcomes were the histological changes of new bone formation in hematoxylin and eosin and Masson trichrome stains, while histomorphometric and radiographic analysis were the secondary outcome followed by statistical analysis (P value was set at .05).

Results

There was a significant increase in the percentage of bone formation in relation to defect height in nanocrystalline compared to microcrystalline form after one month and three months in both histological (p-values of <0.0001) and radiographic results (p-values of <0.0001).

Conclusion

Nanocrystalline calcium sulfate presented a significant enhanced periodontal regeneration compared to the microcrystalline form.

1. Introduction

Periodontitis is a multifactorial chronic inflammatory disease. Periodontal disease (PD) can systemically worsen existing diseases and locally increases the risk of teeth loss.¹ Therefore, deep pockets particularly with unfavorable anatomy, such as the intrabony defect should be diagnosed and treated to avoid dentition depletion. The pattern of bone destruction differs according to disease intensity, type of pathogens, shape and site of bone involved.²

Bone regeneration depends on the synergistic combination of biomaterials, cells, and growth factor therapy to develop a functional PDL. One of most applicable, autogenous, economical bioscaffold used in PDL regeneration is platelet rich fibrin (PRF). It is a second-generation platelet concentrate that forms a 3d scaffold and allows cytokine entrapment and cell migration, with no anticoagulant and contains higher leukocyte content. Several bone grafts had been used in conjunction with PRF.³

Bone graft should be biocompatible, strong enough to preserve space and have low rate of degradation. Calcium Sulfate (CS) was introduced as a filler material and was reported as biocompatible, biodegradable, osteoconductive, angiogenic and hemostatic bone graft. Its natural abundance and the broad industrial and medical applications render CS a highly relevant material in technology.⁴ However, CS has many limitations regarding the regenerative properties such as rapid resorption rate, large surface area and low regenerative capacities.⁵

To overcome these limitations, nano-calcium sulfate (nCS) has been employed as a scaffold in bone regeneration utilizing nanotechnology principles to enhance the physical properties, such as large surface area for growth factor adsorption, controlled rate of releasing the adsorbed material, as well as superior mechanical strength for optimal osteoconductivity and resistance to fractures.⁶

nCS was considered as a viable graft material in bilateral sinus lift augmentation procedures either alone and with PRF⁷ and in critical size defects.⁸ nCS was proposed as promising repair of defects with limited accessibility, such as in periodontal defects,⁸ and was claimed to have clinical and biologic advantages over mCS in fresh extraction sockets.⁶

Searching for cost effective regenerative treatment for pdl is worth-investigating. Herein, this study aimed to evaluate the effect of nCS in periodontal regeneration compared to mCS using PRF as a scaffold, in the dog model. The null hypothesis was that there is no difference in regenerative abilities between the nCS and mCS.

2. Materials, animals and methods

2.1. Study design and grouping

The sample size was calculated using PASS software¹ according to previous studies. Three dogs with split mouth design were used to achieve 84% power to detect a difference of −1.4 between the null hypothesis for which both groups means were 0.9 and the alternative hypothesis for which the mean of group 2 was 2.3 with estimated group standard deviations of 0.7 and 0.4 and with a significance level (alpha) of 0.05 using a one-sided two-sample t-test. One dog was added in each group for possible drop out therefore, the study was conducted on 4 dogs per each group.^9,10

2.2. Animal inclusion criteria

A randomized split mouth design, using eight adult mongrel dogs weighing 18–23 ​kg were selected from Mansoura Experimental Research Center (MERC), Faculty of Medicine, Mansoura University, Egypt. Dogs included in the study were selected with normal dentition, no periodontal disease or history of receiving medication within the preceding month. Dogs with history of systemic, metabolic or immunosuppressive illness, or with missing premolars or molars were excluded. Dogs were pre-acclimatized for one-week prior experimentation. Animals were housed in individual polypropylene cages, under standardized conditions with free access to food. All procedures in the current study conformed to of the ARRIVE guidelines and were approved by the ethical committee, Faculty of Dentistry, Mansoura University (A06091019).

2.3. Surgical procedures

General and local anesthesia were induced using Thiopental Sodium IV (12 ​mg ∕ kg bwt)² and local infiltration of (lidocaine 2% with 1: 80,000 epinephrine).³ To provide enough space for intra-bony defect creation, bilateral mandibular first premolars were extracted prior to experimental surgery and extraction sites were allowed to heal for 2 months.¹¹

After healing, bilateral one wall intra-bony defects (4 ​× ​4mm) were surgically created in the mesial aspect of second premolars.¹² For standardization, Perio Probe PCP UNC- 15 (Hu- Friedy, Chicago, IL, USA)⁴ was used to determine the appropriate width and depth of the defect. In addition, reference notches were created with a small round bur on the root surface at the base of the defect as a guide for histologic evaluation.

The root surface was carefully scaled, planed, flushed with sterile saline, dried with sterile gauze, then bone graft materials were placed on the defect sites where one side was filled with nanocrystalline calcium sulfate (nCS); and the other side received macrocrystalline calcium sulfate (mCS).⁵ PRF membrane was applied as scaffold to cover the graft material. Finally, the flap was closed by simple interrupted suture, using 3/0 absorbable suture.

Platelet rich fibrin preparation: after optimization of anesthesia, during surgical procedures, the lower limb vein blood was collected into a 5 ​mL tube without anticoagulants and immediately centrifuged, according to a previous protocol, at 400 ​g for 10min.¹³

Postoperative care: The animals were maintained on a soft food diet two weeks after surgery. They were given an antibiotic (ceftriaxone injection1gm twice daily for 5 days)⁶ and analgesic (diclofenac sodium, IM, once daily for 3 days).⁷ Oral hygiene was maintained by daily flushing the dog mouth using chlorhexidine 0.12%.¹⁴

24. . Euthanasia of the dogs

Dogs were randomly allocated using list randomizer (https://www.random.org/lists/) into group I, euthanized after one month while group II, euthanized 3 months after graft placement. The euthanasia was done by overdose of thiopental. Standardized digital periapical x-rays were captured for each segment, then the sections were processed for histological evaluation.

25. . Histological evaluation

Sections were fixed, decalcified using a mixture of 250 ​ml formic acid, 100 ​mg sodium citrate and 750 ​ml of distilled water for 5–6 months, then specimens were processed into (5–6 ​μm) paraffin sections, and stained with Hematoxylin and eosin (H&E), and Masson^’s trichrome (MTC) followed by histomorphometric and statistical analysis.¹⁵ (Fig. 1).

Fig. 1.

A schematic diagram based on previous study and an image depicting the landmarks and the parameters used in histometric analysis. CEJ: cementoenamel junction DH: defect height JE:junctional epithelium migration CT: connective tissue adhesion C: cementum NB: new bone regeneration N: base of notch ES: enamel space G: gingiva PD: Periodontal ligament.⁹

2.6. Histomorphometric and statistical analysis

Histometric analysis was obtained using an automated image analysis system⁸ to measure the junctional epithelium, percentage of new bone formed in relation to bone defect histologically and radiographically. Four sections from each block were examined by double-blinded calibrated examiners. Using SPSS (Statistical package for social science) version 25, data were initially tested for normality using Shapiro-Wilk test. The parametric qualitative data were expressed in Mean & standard deviation. Unpaired Student’s t-test. P value ​< ​0.05 was considered statistically significant.

3. Results

3.1. Clinical findings

There was no drop out, no signs of allergy or tissue inflammations suggesting proper conditions and biologically safe materials.

3.2. Primary outcome: histological results

H&E

After one month (group I) revealed newly formed immature bone trabeculae with marked cellularity and wide marrow spaces lined by osteoblasts and progenitor cells in both mCS and nCS filled defects. Both groups revealed PDL fibers, though unorganized, inserted in both cementum and bone (Fig. 2; A&B). In group II, osteons with more organized concentric lamellae were observed in both mCS and nCS filled defects with organized PDL and narrower PDl spaces (Fig. 2; C&D), these results were further confirmed by the radiological results (Fig. 4c).

Fig. 2.

Photomicrograph of whole periodontal structures after 1 month in mCS(A) and nCS(B) (B) treated sides showing the regenerating, though not fully organized pdl fibers, giniva, and new bone trabeculae. The periodontium after 3 months in mCS (C) and in nCS (D) treated sides showing more organized periodontal structures in both groups with signs of calcification and narrowing of PDL space in (c) mCS group. (arrow head ​= ​^osteon), (T ​= ​trabecula), (stars ​= ​bone marrow), (tailed arrow ​= ​PDL), (D ​= ​Dentin), (NB ​= ​new bone). (H&E, x40, Bar ​= ​250 ​μm).

Fig. 4.

() ​A Graph representing percentage of bone formation in relation to bone defect height (relative percentage) both in histological (A) and radiological (B) examination. Test used: Student’s t-test (Unpaired); data were represented in mean and ±sd P ​< ​0.0001. (C) Periapical Radiographs of different groups; mCS after one month (A), nCS after one month (B), mCS after 3 months (C), nCS after 3 months (D).

MTC results

Group I showed varying degree of maturity and positive collagen staining in mCS filled defects. while nCS filled defects showed coalesced osteoid tissue and concentric lamellae arranged in growing osteons with signs of calcification (see Fig. 3A: a&b). In addition to the insertion and formation of PDL fibers in nCS group (Fig. 3B: a1&b1).

Fig. 3.

(A) Masson’s trichrome stain showing variable degree of collagen staining in the whole periodontal structures after 1 month treated with mCS (A) and nCS (B). Periodontium after 3 months treated with mCS (C) and nCS (D). (B) High magnification (400x) for periodontium after 1 month the two studied group treated with mCS (a) and nCS (b).

Group II showed calcified anastomosing bony trabeculae and growing osteons in both mCS and nCS filled defects with varying degree of calcification and more organized PDL inserted in both bone and cementum (Fig. 3A: c&d).

3.3. Secondary outcome: histomorphometric analysis

The amount of newly formed bone was significantly higher in nCS compared to mCS after one month in both histological (29.91 ​± ​0.68; 26.14 ​± ​0.78) and radiological (28.8 ​± ​0.52; 20.8 ​± ​1.51) measurements respectively (p-value of <0.0001). Moreover, the percentage of bone formation in relation to defect height was significantly higher in nCS group compared to mCS after three months (48.35 ​± ​3.52; 22.3 ​± ​2.27) respectively, which was further confirmed with radiographic results (48.96 ​± ​1.2; 21.48 ​± ​0.89) for nCS and mCS respectively. (Fig. 4: A&B ). Similarly, there was a significant decrease in junctional epithelium in nCS compared to mCS after one month (0.6 ​±0 ​.02; 0.95 ​± ​0.11) and after 3 months (0.27 ​± ​0.05; 0.6 ​± ​0.15) respectively with a P value ​< ​0.0001 (Table 1).

Table 1.

Comparison between the two materials used regarding the Junctional epithelium.

	Material				P
	mCS		nCS
	Mean	±SD	Mean	±SD
One Month	0.956	0.12	0.61	0.21	0.0001∗
Three Months	0.63	0.15	0.27	0.06	0.0001∗

Data expressed as mean ​± ​SD.

SD: standard deviation P: Probability ∗:significance <0.05.

Test used: Student’s t-test (Unpaired).

4. Discussion

Dog model is very popular in periodontal regeneration research,¹⁶ due to quite similarity of the size of the teeth, the histological traits and pathophysiological mechanisms of normal as well as diseased periodontium to those observed in human.¹⁷ In addition to reproducible critical-sized defects.¹⁸ We used a one-wall intra-bony box as a clinically relevant defect with minimal self-healing capacities.^12,19

The regenerative role of PRF has been shown in intrabony as well as furcation defects, gingival recession and extraction socket management.²⁰ PRF diminish inflammatory response, attract tissue immune cells and potentiate angiogenesis and collagen formation.²¹ The privilege of nCS results over mCS might be due to the nano-roughness, enhanced adsorption of multiple bone morphogenetic proteins such as osteocalcin (OC) and osteopontin (OP), and the ability to induce more cell proliferation.²²

Similarly Zhang et al., 2016²³ attributed nCS superior bio-compatibility and osteo-conductive properties, compared to mCS, to nanoscale topography, which might have important signaling modalities that control the cell function, such as migration, cell polarization, and cytoskeleton organization. Concomitantly, He et al.²⁴ proposed that injectable nCS paste stimulates bone formation and is an efficient vehicle for stem cells to promote osteogenesis when used in combination with bone morphogenic proteins and mesenchymal stem cells in the critical bone defects of rats.

Park et al., 2011²⁵ reported that nCS granules with (50–500 ​nm) diameter, increased the surface area and consequently enabled greater adsorption of growth factors, higher surface area for attachment of osseous cells and more efficient osteoconductivity, in addition to controlled degradation of the material (over 10–12 weeks),²⁵ while mCS was within 4–6 weeks¹⁰ Moreover, they reported that nCS was harder than mCS, using surface microhardness testing, proposing an additional advantage to the scaffolding properties of nCS.²⁵

Additionally, another study compared mCS and 2 different nCS both radiologically and clinically and attributed lesser bone gain to rapid degradation of mCS, that might explain the decreased bone percentage in three-month group both histologically and radiographically.²⁶

In contrast to the finding of the present study, Kim et al., 2006,²⁷ reported that CS showed inflammatory cells around borders in connective tissue without formation of osteoid bone. Furthermore, Hong et al., 2008,⁹ claimed that regenerative properties of CS get better when combined with alloplast, compared to the CS alone. While Jain et al., 2012,²⁸ and Kumaria et al., 2014,⁶ reported no significant difference between CS and nCS in socket preservation before dental implant placement.

This study was the first to compare between nCS and mCS at histological level regarding periodontal regeneration. However, other studies reported using other nanoparticles bone graft such nanohydroxyapetitie in regarding periodontal regeneration.^29,30

This study was conducted in parallel with a clinical study (Could nanocrystalline calcium sulfate bone graft maximize the regeneration of periodontal intrabony defect? ”unpublished data”) to investigate the effect of nCS and mCS on bone regeneration in intra-bony defect at histological level. Based on the finding of this study, it can be concluded that microscopic and radiographic findings proposed nanocrystalline calcium sulfate as a cost-effective alternative to conventional microcrystalline form of calcium sulfate in periodontal tissue regeneration regarding new bone formation and cellular proliferation.

The combination of nCS with different platelet concentrates is a forthcoming interest.