The Use of Angelica Sinensis and β-tricalcium Phosphate in the Management of Surgically Induced Critical-sized Class II Furcation Defects in Dogs

doi:10.21203/rs.3.rs-1717123/v2

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The Use of Angelica Sinensis and β-tricalcium Phosphate in the Management of Surgically Induced Critical-sized Class II Furcation Defects in Dogs

https://doi.org/10.21203/rs.3.rs-1717123/v2

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Objective: Evaluating the effectiveness of Angelica sinensis, a widely used traditional Chinese medicine, in conjunction with Beta-tricalcium phosphate (β-TCP) bone graft in the management of induced critical sized class II furcation defects in adult healthy mongrel dogs.

Materials and Methods: A randomized study design was conducted on the third and fourth premolars of six healthy mongrel dogs. A total of twenty-four class II furcation critical-sized defects were surgically created. After reflecting a fully-reflected mucoperiosteal flap, twelve defects were filled with Angelica sinensis powder mixed with Beta-tricalcium phosphate (β-TCP), then collagen membranes were applied over the defects (experimental group). The other twelve defects were filled with β-TCP followed by collagen membrane application (control group). The dogs were sacrificed at the end of fourth and eighth weeks. Jaw segments containing the operated teeth were dissected and processed for histological examination and morphometric analysis of the regenerative features.

Results: The histological results revealed that bone formation and regeneration of cementum and PDL progressed more in experimental groups than in control groups. Moreover, the regenerative features were more advanced in the second observation period. The histomorphometry revealed that the surface area of newly formed bone and the thickness of its trabeculae were higher in the defects treated with Angelica sinensis and β-TCP than in only β-TCP-filled defects at both observational periods.

Conclusions: Angelica sinensis has accelerated the regenerative potential of the interradicular bone and has enhanced the effect of β-TCP for interradicular bone regeneration in dogs.

Angelica sinensis

regeneration

bone graft

furcation defects

The periodontal disease is the most common infectious oral condition[1]. It is a complex, multifactorial, polymicrobial infection involving the destruction of tooth-supporting tissues including alveolar bone, periodontal ligament and cementum [2]. Furcation involvement is the result of pathologic resorption of the supporting alveolar bone caused by the progression of periodontal disease. The treatment of such defects always represents a clinical challenge [3].

Periodontal regeneration is the restoration of the tooth-supporting tissues structurally and functionally. Although a gain of clinical attachment level and bone fill are clinical indicators for successful regeneration, it is only by histological means the conclusive evidence of true regeneration can be achieved [4].

Bone grafting and barrier membranes are used together with biologically engineered tissue inductive proteins (e.g., growth factors, extracellular matrix proteins and bone morphogenetic proteins) resulting in osseous and periodontal regeneration [5].

Several bone grafting materials have reported to induce cell and bone tissue in-growth at the material surface [6]. Examples of commonly used grafts are autogenous grafts harvested from the patient's iliac crest, mandibular ramus, or other intraoral sites, freeze‐dried bone allograft, demineralized freeze‐dried bone allograft, xenograft obtained from equine, porcine, or bovine bone, synthetic calcium sulphate, calcium phosphate, β tricalcium phosphate, hydroxyapatite and composite bioceramics [7, 8].

Barrier membranes act as a physical barrier for epithelial cell exclusion and for periodontal ligament and alveolar bone cells to repopulate the defect, thus allowing the desired regeneration of periodontal tissues [9]. The first generation of barrier membranes were the non-resorbable membranes polytetrafluoroethylene (PTFE), titanium reinforced ePTFE, high-density-PTFE, or titanium mesh [10]. In order to eliminate the need for second surgical procedure, the synthetic resorbable barrier membranes polyesters (e.g., polyglycolic acid (PGA), polylactic acid (PLA), polycaprolactone (PCL) and natural barriers as tissue-derived collagen-based membranes from human skin, bovine Achilles tendon or porcine skin have been developed as second-generation membranes [11, 12].

Complementary and alternative medicine (CAM) is considered a widely different group of approaches used to prevent or treat diseases [13]. Barnes, et al.[14] has defined CAM as "a group of diverse medical and health care systems, therapies, and products that are not presently considered to be conventional medicine ". Chinese herbal medicine has a history of more than 2,500 years and has accomplished remarkable effects in the clinical practice [15, 16]. The therapeutic approaches in typical Traditional Chinese Medicine include herbal therapy, acupuncture and dietary therapy [17].

Angelica sinensis herb has been used to increase bone formation due to its both osteogenic and angiogenic effects[18]. Moreover, Angelica sinensis polysaccharide may stimulate the osteogenic differentiation of rat bone marrow mesenchymal stem cells and promote bone regeneration under high glucose conditions by activating the Wnt/β-catenin signaling pathway [19]. Gao, et al.[20] provided the first evidence that Ferulic acid (FA) from Angelica sinensis can enhance the proliferation and differentiation of osteoblasts in vitro by expressing a number of osteogenic genes.

The aim of this paper is to investigate the regenerative potential of Angelica sinensis in periodontal defects.

The research protocol was approved by the institutional experimentation and Animal Ethical Committee of Alexandria University, (IRBNO:00010556‐IORG0008839). It is a preclinical animal study and conformed to the updated ARRIVE 2.0 guidelines.

Six mongrel dogs (Canis familliaris) with inclusion criteria of being systemically healthy male dogs, 18-24 months old and weighing approximately 10- 18 Kg are included in the study. The dogs are matched regarding; defect dimensions, sex, age, weight, type of diet, and environmental housing conditions. Exclusion criteria are dogs included in previous experimental study and dogs with any apparent illness or wounds.

Grouping: The study included twenty-four surgically induced critical sized class II furcation defects in the buccal surface of the right and left mandibular premolars (P_3,P₄). Twelve defects were assigned in the experimental group and were managed using Angelica sinensis*, ß‐TCP allograft† and collagen membrane‡. The other twelve defects acted as the control group with ß‐TCP and collagen membrane only. Angelica sinensis is supplied in 1 bottle of 100 g of concentrated granules extracted from 330 g of the raw herbs. The size of the granule ranges from 3 to 4 μm. In order to minimize the risk of cofounding, random allocation was done, by using a computer-generated random sequence of numbers to assign treatment status. Randomization was done by giving each defect used in this study a number from 1 to 24 and using computer assisted software, a number of 12 defect was selected in each group

Surgical procedure

Proper sterilization of all the surgical instruments used in the procedures was ensured. The animals were generally anesthetized by sodium thiopental§ intravenous injection (13 mg/kg).1:100,000 epinephrine. Sulcular incisions at the buccal aspect of the mandibular third (P₃) and fourth (P₄) premolars in the right and left quadrants were then followed. Mucoperiosteal flaps were fully reflected. A rotary 0.5 mm rounded carbide bur was used to create Grade II critical sized furcation defects of 3 × 4 at P3 and P4 for each dog [21, 22]. The inter-radicular bone was removed under copious irrigation using sterile saline.

For the future histological examination, a reference point of the bone level surrounding the created defects at the time of the surgery was essential[23]. Root planing by universal curette and root conditioning using Ethylenediaminetetraacetic Acid (EDTA)ǁ were carried out to the exposed root surfaces. The defects of control group were filled with β‐TCP alone, while defects of the experimental group were filled with Angelica sinensis granules mixed with β‐TCP (1:1). Trimming of the collagen membranes to the proper size extending 2-3 mm all around the created defects was followed by the application of the membranes to cover the graft filled defects. The flap was repositioned coronally to have a complete coverage of the defect area. The wound closure was achieved with simple interrupted suturing (Figure 1)

Postoperative care

For the following week, the dogs received the medication mixed with their food. Throughout the study period, the dogs were fed soft diet to minimize the possibility of local trauma at the site of operation. Sutures were removed after 10 days.

Animal euthanasia

Three dogs were euthanized at 1 month postoperatively and the other three dogs after 2 months. The euthanasia involved the use of overdose intravenous injection of concentrated thiopental sodium⁴.

Histological procedure

After euthanasia, the mandibles were dissected out and the jaw segments containing the operated teeth were separated and fixed in 10٪ neutral buffered formalin. They were decalcified and processed to obtain 5 microns thickness mesiodistal serial sections which were stained with Hematoxylin and Eosin (H&E) and Gomori trichrome and examined by light microscope to evaluate the regenerative potential in the defects and perform the morphometric analysis.

Histomorphometric quantitative analysis

Histologic photomicrographs were quantitatively analyzed using image J 1.46 r software [24]. Measurements were preformed in mm or fractions of mm.

Twovariables were analyzed:

Mean percentage of the newly formed bone surface area. Standardized magnification of x100.
Thickness of newly formed bone trabeculae in mm. Standardized magnification of x100.

From each of the 24 specimens of the defects, two standardized depths were selected from which two sections were obtained and hence a total of 48 sections were examined (24 sections from the control group ,12 for each observation period and the other 24 sections from the experimental groups, 12 for each observation period). From each of the chosen sections, one standardized image for specific same structures needed for quantification was taken then measurements were recorded and statistically analyzed.

All the morphometric analysis was carried out by one investigator. Each measurement was taken 2 times, two days apart, and the mean was then recorded.

Statistical analysis

Data obtained from the histomorphometry were statistically analyzed. Quantitative data were analyzed using median, mean ± standard deviation and range (minimum -maximum). The level of Significance of the results was calculated at the 5% level. Comparison between the results of the study and experimental groups was done using two-tailed students t-test (GraphPad software.Inc.).

* Active Herb company, Suite E, San Diego

† Med bone Biomaterials Sentra, Portugal.

‡ Bioimplom GmbH, Giessen, Germany

§ Sandoz GmbH Biochemiestraße, Österreich, Austria

ǁ META BIOMED CO, LTD, Korea

Clinical observations

During the postoperative period, no clinical adverse reactions were noted. The dogs were clinically healthy and did not exhibit any sign of site infection or wound contamination.

Histological results

4 weeks observation period:

In the experimental group formation of a good amount of regenerative bone was noted in most of the sections obtained from different samples of experimental group. However, the formed bone did not fill the defects completely, but some areas in the central regions accommodated β-TCP particles with some patches of Angelica sinensis, (Figures 2A and 2B). Active figures of Angiogenesis were traced in the regenerating PDL, (Figure 2C). At higher magnification of examination, prominent cementoblast cells were traced on the border of the forming cementum and were separated from it by a layer of cementoid, (Figure 2D). Also, voluminous osteoblasts could be traced bordering the newly formed trabeculae, (Figure 2E). In trichrome stained sections, the regenerating fibers of the forming PDL could be traced inserting in the forming alveolar bone, (Figure 2F).

In the control group, the histological picture differed from that of the experimental group with less amount of formed bone which was especially deficient at the most coronal boundary of the defect. The regenerating bone consisted of irregular thin trabeculae with intervening spaces containing considerable amount of β-TCP particles and less blood vessels than in the experimental group, (Figures 3A and 3B). Towards the periphery of the defect, the forming fibers of the PDL appeared thin, discontinuous, and exhibited random course for insertion in either of the regenerating alveolar bone or cementum, the latter was bordered by flattened cementoblasts. These randomly arranged fibers enclosed flattened fibroblasts, (Figures 3C and 3D).

8 weeks observation period:

The histological findings in the experimental group were the best among all of the included groups in this study. Bone formation extended to a higher level coronally than its level at the 4 weeks observational period. The formed bone filled almost most of the defect, (Figure 4A). The formed trabeculae were thicker than those seen in the 4 weeks observation period and exhibited an outstanding fashion of communication. A considerable density of blood vessels was traced among these trabeculae, (Figure 4B). In this group, the regenerating bone characterized by outstanding feature of remodeling revealed by different levels of bone stainability. The remodeling figures predominated and occurred in closed association with patches of Angelica sinensis (Figure 4C). Adjacent to both of the regenerating cementum and bone, the cementoblast and osteoblast cells appeared sizable and highly organized in relation to either tissues than their appearance in association with the other groups, (Figures 4D and 4E). In trichrome stained sections, the PDL fibers formed thick bundles and could be traced for deeper levels in the regenerating alveolar bone and surrounded by many blood vessels, (Figure 4F).

In the control group, the defects exhibited moderate degree of filling with bone trabeculae of variable size and separation. Moderate figures of blood vessels and β-TCP particles were seen among these trabeculae, (Figures 3E and 3F) Towards cementum, Collagen fibers of regenerating PDL exhibited strong appearance of remodeling and thickness variation. Cementum reformation and regeneration were also evident, (Figure 3G). Collagen fibers of regenerating PDL were inserted in the regenerating bone, but most of them did not exhibit the bundle aggregation which was seen in the experimental group of the same observational period, (Figure 3H)

Results of histomorphometric analysis

The histomorphometric assessment of the three measured parameters (height of the formed interradicular bone, percentage of bone surface area, and thickness of bone trabeculae) at the two observation periods showed comparable results in both groups with greater values in Angelica sinensis study group. ( table 1)

The percentage of bone surface area of the Angelica sinensis / ß‐TCP experimental group was significantly higher than that in the control group after 1 month (mean = 58.88±13.92 and 44.63±7.94 % respectively) with P=0.002 and after 2 months (mean = 78.35±5.76 and 64.70±7.75% respectively) with P=0.02.

After 1 month the newly formed trabecular bone thickness in the experimental group was significantly higher than that in the control group (mean =0.468±0.08 and 0.23 ±0.05mm respectively) with P=0.0001. The difference was also significantly higher at 2 months period (mean=0.57±0.11 and 0.37±0.05mm respectively) with P= 0.0001.

This study was conducted to investigate the osteogenic potential of Angelica sinensis, a well-known traditional Chinese herb, when used in the management of surgically induced class II furcation defects. Randomization of the induced defects on whether to receive Angelica sinensis and β‐TCP or β‐TCP alone has excluded the environmental and biological tissue factors that are present between different animals. Any difference between the groups could therefore be referred to the regenerative effect of Angelica sinensis. Few if any study has measured its regenerative osteogenic effect when used intraorally on large-sized animal (dogs). To the best of our knowledge, this study is the first one to include the application of Angelica sinensis in the management of class II furcation defects in experimental animals. The literature does not contain similar studies on human.

The results obtained in this study have rejected the null hypothesis which was proposed before preforming the experiment.

The quantitative analysis of the current study has reflected the osteo-inductive potential of Angelica sinensis. This was also revealed in an experimental study by Liao F, et al.[19] in which Angelica sinensis Polysaccharide promoted the osteogenic differentiation of rat bone marrow mesenchymal stem cells under high glucose culture through the activation of the Wnt/β-catenin signaling pathway, thus inducing bone regeneration. This osteogenic differentiation effect was further confirmed by Xie X, et al.[25] in which Angelica polysaccharide has promoted the same effect on rats mesenchymal stem cells by regulation of long non-coding RNA H19. This in vivo study supported the promoting action of angelica sinensis on bone formation.

Another in vitro study had also demonstrated that ferulic acid, an active component of Angelica sinensis, promoted proliferation and differentiation of osteoblast cells and has enhanced osteogenic genes expression [26].

The histological results of the current study have strongly elucidated the bone regenerative capability of Angelica sinensis. This could be supported by various studies which proved that Angelica sinensis can inhibit wear debris particles-induced inflammatory bone resorption in mice[27] and can suppress osteoclastogenesis through inhibiting RANKL-mediated osteoclast differentiation in bone marrow macrophages decreasing bone resorption and preventing bone loss [28, 29] .

Moreover, the high regenerative ability of Angelica sinensis can be contributed to its anti-inflammatory effect which was proved by Kim YJ, et al.[30] who reported that Angelica sinensis root water extract has anti-inflammatory effect on lipopolysaccharide (LPS)-induced RAW 264.7 mouse macrophages. Other studies reported that the volatile oil of Angelica sinensis has shown a potential anti-inflammatory mechanism by plasma metabolomics approach that has significantly inhibited systemic inflammatory response mediated by acute local stimulation[31, 32]

Another mechanism that can explain the current resulted effects of Angelica sinensis is its antioxidant property which was proved by the study of Tian S et al.[33] in which Angelica sinensis polysaccharides were extracted using the ultrasound-assisted extraction method. Another in vivo study proved that this antioxidant activity protects rat chondrocytes from apoptosis induced by hydrogen peroxide which makes it accepted to be used as a therapeutic supplementation for the treatment of osteoarthritis[34].

Apart from the previously reported strong stimulation of osteogenesis, the present study proved that Angelica sinensis exerted a regenerative effect on cementum and PDL regeneration. The histological sections of the experimental groups showed increased cementoblast-like cells along the uniformly deposited cementum layer into which Sharpey’s fibers were inserted and fibroblast-like cells in a highly vascular PDL space. The thick fiber bundles of PDL in experimental group of 8 weeks reflects the stimulating effect of Angelica sinensis on fibroblast cells. These results are in accordance with the results of studies by Zhao H et al.[35, 36]in which SBD.4A-a defined multicomponent preparation of Angelica sinensis has stimulated inflammatory healing responses mediated by human periodontal ligament cells, through the proliferation of fibroblast cells which in turn increased the amount of produced hyaluronic acid.

Another considerable finding observed in the histological sections of Angelica sinensis test groups were the greater density of newly formed blood vessels as compared to the control groups. Angelica sinensis exerted an evident effect on the proliferation and dilation of blood vessels revealed by the close association between them in most of the sections examined from the two experimental groups. The current study results are in agreement with the results of a study which proved the angiogenic potential of Angelica sinensis and described how this angiogenic potentiality of Angelica sinensis can treat ischemic strokes [37]. Meanwhile, the investigation by Lam HW et al.[38] revealed this angiogenic effect on human endothelial cells (HUVEC) in vitro and zebrafish in vivo.

When comparing different modalities used in the treatment of Class II furcation defects in dogs, Angelica sinensis shows comparable results as regard to percentage of bone surface area. Deliberador TM et al. [39] investigated the effect of autogenous bone graft with or without a calcium sulfate barrier. After three-month observation period, the percentage of bone surface area for autogenous bone graft with and without a calcium sulfate barrier were 59.85(±20.90) and 64.95(±15.75) respectively whereas for Angelica/ β-TCP was 58.88(±13.92) after 2-month observational period only. Another study by Simsek et al. [40] comparing the effect of mesenchymal stem cells (MSCs) and autogenous cortical bone graft (ACB) in the treatment of class II furcation defects in dogs revealed percentage of new alveolar bone area after 8 weeks of 84.60 (±4.85) for ACB and for 80.47 (±8.23) for MSCs . These results are comparable to the result obtained in the current study in which the percentage of the formed bone surface area after 8 weeks was 78.35 (±5.76)

Through the study model in this investigation, Angelica sinensis has demonstrated increased regenerative capability through measuring the amount of the formed bone. In further researches, it is recommended to analyze the surface area of the proliferating blood vessels. Furthermore, adjusting the ratio of Angelica sinensis to β-TCP to be 2:3 or using perforated collagen membrane instead of the occlusive type to allow the migration of gingival stem cells to the defect can be assessed.

The management of furcation defects with Angelica sinensis in conjunction with β-TCP leads to better bone formation as regard to the rate, quality and quantity of new bone when compared to the use of β-TCP alone, Angelica sinensis, a biocompatible material with a highly regenerative potential, enhances the effect of β-TCP in bone regeneration and accelerates the regeneration potential in class II furcation defects in dogs.

ACKNOWLEDGMENTS

The authors would like to express their appreciation to Alexandria University for supporting the study. The authors reveal no conflicts of interest regarding this study.

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Table 1 is available in the Supplementary Files section.

No competing interests reported.

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The Use of Angelica Sinensis and β-tricalcium Phosphate in the Management of Surgically Induced Critical-sized Class II Furcation Defects in Dogs

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