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Table of Contents   
ORIGINAL RESEARCH  
Year : 2019  |  Volume : 30  |  Issue : 4  |  Page : 558-567
Calcium sulfate-based bioactive cement for periodontal regeneration: An In Vitro study


1 Division of Bioceramics, Biomedical Technology Wing, Sree Chitra Thirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala, India
2 Division of Tissue Culture, Biomedical Technology Wing, Sree Chitra Thirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala, India

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Date of Submission06-Jan-2018
Date of Decision06-Feb-2019
Date of Acceptance30-May-2019
Date of Web Publication18-Nov-2019
 

   Abstract 


Background and Objective: Various types of osteoconductive graft materials are used for the management of alveolar bone defects arising out of periodontal disease. Inorganic, self-setting, bioactive bone cements are suggested to be most appropriate because they can conformally fill the bone defect and resorb progressively along with the regeneration of the host site. A new calcium sulfate-based bioactive bone cement (BioCaS) is developed, having simplicity and effectiveness for bone grafting applications. The response of primary human periodontal ligament (hPDL) cells to this material is investigated through in vitro cell culture model so as to qualify it for the repair of periodontal infrabony defects. Method: The BioCaS was designed as powder-liquid combination with in-house synthesized high purity calcium sulfate hemihydrate incorporating hydrogen orthophosphate ions. hPDL cells were isolated, cultured and characterized using optimized primary cell culture techniques. The cytotoxicity and cytocompatibility of the BioCaS samples were evaluated using the hPDL cells, with hydroxyapatite ceramic material as control. Osteogenic differentiation of the hPDL cells in presence of BioCaS was also evaluated using Alizarin red staining, Alizarin red assay, Von Kossa staining and Masson's trichrome staining. Results: The primary cell culture techniques yielded a healthy population of periodontal ligament cells, with fibroblast morphology and characteristic marker expressions. The hPDL cells exhibited good viability, adhesion and spreading to the BioCaS cement in comparison to sintered hydroxyapatite. In addition, the cells differentiated to osteogenic lineage in the presence of the BioCaS cement, without extraneous osteogenic supplements, confirming the inherent bioactivity of the cement. Conclusion: The new BioCaS cement is a potential candidate for the repair of periodontal defects.

Keywords: Alveolar bone grafts, BioActive calcium sulfate cements, biomaterial-cell interactions, periodontal ligament cells

How to cite this article:
Das EC, Kumary T V, Anil Kumar P R, Komath M. Calcium sulfate-based bioactive cement for periodontal regeneration: An In Vitro study. Indian J Dent Res 2019;30:558-67

How to cite this URL:
Das EC, Kumary T V, Anil Kumar P R, Komath M. Calcium sulfate-based bioactive cement for periodontal regeneration: An In Vitro study. Indian J Dent Res [serial online] 2019 [cited 2019 Dec 15];30:558-67. Available from: http://www.ijdr.in/text.asp?2019/30/4/558/271048



   Introduction Top


Periodontal disease is a matter of serious concern in dentistry because it can destroy the integrity of “periodontium” and eventually lead to tooth loss. The “periodontium” is a complex tissue structure consisting of the alveolar bone, the cementum covering the tooth root, the periodontal ligament connecting the cementum and supporting alveolar bone and the gingiva. This structure provides sensory perceptions and absorbs, and distributes the occlusal masticatory forces.[1] Periodontal diseases progressively destroy the collagen fibers constituting the periodontal ligament leading to loss of attachment between the supporting alveolar bone and cementum and often cause alveolar bone loss resulting in tooth mobility. In the advanced stages, loss of alveolar bone occurs with the formation of “periodontal pocket” around the tooth which vary from one-walled to three-walled defects with furcation involvement.[1],[2]

The treatment approach at the initial stages of a periodontal disease or infection is to debride the affected site, enabling a natural wound healing response.[1] The fibrin in blood clot will act as a matrix and a reservoir of growth factors responsible for the periodontal ligament cell repopulation and regeneration.[3],[4] The periodontal ligament regeneration is characterized by the formation of new, healthy ligamental attachment between cementum and alveolar bone through periodontal ligament (PDL) cell migration and repopulation between alveolar bone and cementum.[2],[3] However, in most cases, epithelial interference from the gingiva hinders the natural healing process. As a remedial measure, “barriers” (made of biocompatible and resorbable materials, made in the form of membranes) could be placed in between the gingiva and the debrided tooth.[5] The technique is known as “guided tissue regeneration” (GTR), which helps to achieve better and predictable regeneration of the lost periodontium, compared with conventional debridement.[5]

A substantial alveolar bone loss calls for osseous grafting in addition to GTR. Synthetic osteoconductive bone graft materials (alloplasts) are preferred due to their off-the-shelf availability and the capacity to integrate with the host bone and to participate in the remodeling of the defect.[6],[7] Bone grafting is now in wide practice, yet the combined use of barrier materials and grafts makes the procedure very complex and often very expensive.[8] Instead, another approach could be taken to mobilize the remaining healthy progenitor cells to the site of healing and thereby to promote endogenous regeneration.[9] The use of autologous platelet concentrates like platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) has been suggested.[10] Good clinical results were observed with PRP gels and more evidences are available on the use of PRF. However, the procedure of using platelet concentrates failed to prove true relevance in regular practice because it requires mildly invasive collection of blood and availability of chair-side centrifuge to isolate PRP and PRF. Affordability also is a problem because the procedure is expensive and time-consuming. Moreover, the use of platelet concentrates does not obviate the need for barrier membranes.

A notable and hopeful innovation in periodontal regeneration is the use of moldable, resorbable and self-setting cements which have bioactive properties. Such cements will serve the purpose of an osteoconductive graft providing conformal filling at the defect site. At the same time, they can be contoured and solidified over the region, thereby obviating the need for a barrier to prevent the epithelial cells migrating to the site. Thus bioactive self-setting cements can act like a “barrier-graft.” Calcium phosphate cement (CPC) has been identified as a promising candidate in this category.[11]

In a recent development, biocompatible cement based on calcium sulfate has been developed which is as efficacious as CPC.[12],[13] The new cement material incorporates hydrogen orthophosphate groups in medical grade calcium sulfate hemihydrate, and undergoes hydraulic setting pathway like gypsum cements. The material, named “Bioactive calcium sulfate (BioCaS) cement,” is found to be safe for in vivo use and showed healing of experimental bone defects in an animal model.[13] It possesses the simplicity of calcium sulfate cement while retaining the bioactive properties of calcium phosphate cement. Being a candidate “barrier graft” material, it is interesting to explore the potential of BioCaS to heal periodontal defects.

The present paper investigates the cytocompatibility and in vitro osteogenic differentiation of human Periodontal Ligament cells (hPDL cells) in presence of BioCaS cement. The control material chosen was hydroxyapatite porous ceramic material because it is the established alloplastic bone graft material for periodontal grafting.[11]


   Materials and Methods Top


Preparation of the Bioactive Calcium Sulfate (BioCaS) cement

The powder part of BioCaS cement has been made in house from high purity ingredients (all purchased from M/s Merck, India). Calcium sulfate (in dihydrate form) was first synthesized from analytical grade Ca(NO3)2.4H2O (98 wt% purity) and concentrated H2 SO4(97-99 wt% purity) through a “drowning-out” wet precipitation technique using isopropyl alcohol (99.5 wt% purity), the method of which has been described elsewhere.[12] The resultant precipitate was isolated through centrifugation and freeze drying, which contained uniform low-dimensional (3-5 μm) rod-shaped crystals of calcium sulfate, when observed in scanning electron microscopy (SEM).[12] It was heated for 18 h at 120°C at the atmospheric pressure in an oven to obtain calcium sulfate hemihydrate (the beta phase). Disodium hydrogen orthophosphate dihydrate (99.5 wt% purity) was added to this powder in a proportion 2% w/w to make the BioCaS cement.[13],[14] On wetting with deionized distilled water (0.5 ml per gram), the powder will give a putty which will set in 12 min. The physicochemical characterization of BioCaS cement has previously been reported.[12],[13],[14]

The samples of BioCaS cement were prepared by filling the cement putty in prefabricated silicone molds with cavities of 10 mm diameter and 2 mm height. The cement discs were taken out after setting, washed in distilled water followed by drying for 24 h, and sterilized using ethylene oxide for the experiment. The control samples for the osteogenic experiment, hydroxyapatite (HA) ceramic discs, were made using in-house prepared high pure powder obtained through chemical precipitation. The calcined fine particles of 125 μ grade were taken in a quantity of 500 mg, in a die system of 12 mm diameter and made into discs by compressing with the help of a hydraulic press. These were sintered at 1100°C for 2 h to obtain HA discs of about 10 mm diameter. In the finishing step, the discs were polished to bring the thickness to 2 mm, cleaned in distilled water and sterilized by autoclaving.

Isolation and characterization of periodontal ligament cells

The hPDL cells were isolated, characterized, and evaluated for the osteogenic differentiation potential. The study was done based on a protocol approved by the Institutional Ethics Committee of Sree Chitra Tirunal Institute for Medical Sciences and Technology, (SCTIMST) Thriruvananthapuram, India.

Cell isolation was done from discarded, caries-free permanent teeth, extracted for orthodontic reasons, collected through ethical route. Teeth were transferred immediately after extraction to phosphate-buffered saline (PBS) containing antibiotics (0.1% penicillin/streptomycin, and 0.25 μg/ml fungisone) kept in aseptic vials. The samples were transported to cell culture lab and subsequent steps were performed under aseptic conditions inside a biosafety cabinet (Esco, Singapore). The teeth were repeatedly washed with PBS (containing antibiotic and antifungal agents) to remove surface debris. The periodontal ligament tissue was scraped off from the tooth root in a corono-apical direction from the middle third to the apical third of the root. The tissue fragments were minced to 1-2 mm pieces and treated with trypsin (0.25% kept at 37°C) for 5 min in a  Petri dish More Details. After enzyme treatment, the tissue fragments were incubated with minimal essential medium, alpha modification (MEMα; Gibco) containing 10% fetal bovine serum (FBS; Gibco) and 100 IU penicillin/streptomycin (Gibco) in a CO2 incubator (Sanyo, Japan) set at 37°C having a humidified atmosphere of 5% CO2. The cells migrating out from the explants were monitored under an inverted phase-contrast microscope (Nikon TS100, Japan). The cells were characterized and passaged on achieving the confluent state. In this step, the confluent, adherent cells were detached using 0.25% trypsin (Gibco) at 37°C for one minute, transferred to fresh cell culture bottles and subcultured. Cells at passage 2-3 were used for experiments.

The hPDL cells at passage 1 were characterized by immunofluorescence staining for the expression of intermediate filament marker (Vimentin), mesenchymal stem cell markers (CD90, CD105, and Stro1) and ligament specific marker (Scleraxis, Abcam primary antibodies).[15] The hPDL Cells were seeded onto 1 cm 2 glass coverslips and at 48 h the medium was discarded, the cells were rinsed with PBS and were fixed with 4% paraformaldehyde (PFA) for 1 h. After fixation, washing was done thrice with PBS and permeabilized with TritonX100. Nonspecific binding of antibodies was blocked by incubating with 1% BSA for 10 min. The cells were treated with the primary antibodies at 4°C overnight. After rinsing with PBS, the cells were treated with secondary antibody (anti-rabbit AlexaFluor 546 for scleraxis and anti-mouse AlexaFluor 488 for all other primary antibodies (Abcam) and kept for 1 h at room temperature under dark. The cell nucleus was counter stained with Hoechst 33258 (5 μg/ml, dissolved in PBS) and samples were observed under a fluorescence microscope (Leica DMI 6000, Germany) equipped with filters suitable for green (I3) and red (N21) emission.

Cytotoxicity

The cytotoxicity of BioCaS was analyzed with hPDL cells through direct contact method. The cells were seeded on 24 well plates at a density of 3 × 104 cells/well and maintained in alpha MEM for 24 h. The sample and control discs [BioCaS and sintered hydroxyapatite (HA)] were carefully placed on the cells and incubated for 24 h, 7 days and 14 days. The cell response was analyzed under a microscope (Nikon TS100, Japan) and the cytotoxicity was graded from 0 to 4 (0 - no cytotoxicity, 1 - slight cytotoxicity, 2 - mild cytotoxicity, 3 - moderate cytotoxicity, and 4 - severe cytotoxicity) based on the morphology, cell lysis, cell detachment and vacuolization of the cells around the material.

Cytocompatibility

The cytocompatibility was analyzed by evaluating cell adhesion and cell viability on BioCaS material. HA, being an accepted osteoconductive material, served appropriate comparison. The cells in passage 2 were seeded onto BioCaS cement discs and sintered HA discs (10 mm diameter and 2 mm thickness) and cultured in alpha MEM, 10% FBS, 100IU penicillin/streptomycin for 48 h. The cells were analyzed for viability, adhesion and morphology.

Viability of cells was determined with a combination of flourescein diacetate (FDA; Sigma, India) and propidium iodide (PI; Sigma, India).[16] The cells were incubated with live-dead solution containing FDA (5 μg/ml in serum free MEM) and PI (0.5 μg/ml) for 5 min and observed under fluorescence microscope (Leica).

Actin cytoskeleton of the cells was stained with Actin green 488 (Molecular probes, Thermo Fischer Scientific) at 48 h. The samples were seeded with hPDL cells, cultured for 48 h and was fixed with 4% PFA (1 h), washed thrice with PBS and permeabilized with 0.1% Triton X100 (5 min). Cells were treated with 1:100 dilution of Actin green 488 (Molecular probes, Thermo Fischer Scientific) for 30 min at room temperature and was counterstained with Hoechst 33258 (0.5 μg/ml) for the nucleus. Cell adhesion and cytoskeletal distribution was analyzed under fluorescence microscope (Leica, Germany).

As a supporting investigation, SEM was done separately on the BioCaS discs (HA discs were not included in this experiment). Cells adhered on BioCaS was fixed with 2.5% gluteraldehyde solution and dehydrated in an ascending series of ethanol (30%, 50%, 70%, 90% and 100%). The dehydrated samples were dried in a critical point drying machine and then sputter coated with gold for SEM imaging.

Metabolic activity

The metabolic activity of hPDL cells after direct contact test was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reduction assay.[17] The cells were seeded in 24-well culture plates at a density of 3×104 cells/well and cultured for 24 h. Then the test materials (BioCaS discs and sintered HA discs) were placed gently over the cells. The hPDL cells cultured without test materials were considered as control and medium alone was taken as the blank control. After incubating the cells with and without the test samples for 24 h, the culture medium was replaced with 200 μl of freshly prepared MTT solution (1 mg/ml) in serum-free αMEM and incubated for 2 h in the dark. The MTT solution was then discarded and the intracellular formazan crystals, formed by the action of dehydrogenase enzymes in metabolically active cells in each well, were viewed and dissolved in 200 μl of isopropyl alcohol. The formazan crystals formed was quantified colourimetrically by measuring the absorbance at 570nm in a microplate reader (BioTek USA). The optical density (OD) obtained was used to calculate the percentage viability of hPDL cells in the test compared with the cell control.

Osteogenic differentiation of hPDL Cells in the presence of BioCaS cement

The cells at passage 3 were used for the osteogenic differentiation. The hPDL cells were trypsinized and seeded onto 12-well plates, at a density of 104 cells/cm 2, and the test materials (BioCaS cement discs and sintered HA discs) were carefully placed over the cells. The culture was done in the presence of regular cell culture medium containing αMEM, 10% FBS, and 100 IU penicillin/streptomycin. Being a study of osteogenic property, no inducing agents or supplements were introduced in the case of test samples. The hPDL cells cultured in osteogenic induction medium was considered as positive control and the cells cultured in regular medium was used as negative control. The following analyses were done to confirm the osteogenic property of BioCaS material.

Alizarin red staining

Alizarin red is a calcium chelating dye used to detect calcium deposits in in vitro osteogenic cell culture experiments. The hPDL cells were fixed on 7, 14, and 21 days using 4% PFA for 1 h, washed thrice with deionised water, and treated with 2% alizarin red (Sigma Aldrich) solution for 20 min. The stained samples were washed with deionized water and viewed under microscope (Nikon TS100, Japan).

Alizarin red assay

The mineralization was quantitatively analyzed on 21st day by Alizarin red assay. The Alizarin red stained samples were rinsed with deionized water, the retained dye was solubilized in 90% alcohol and transferred to 96-well plate for the quantitative analysis by colourimetric assessment at an absorbance of 505 nm in a multiwell plate reader (BioTek, USA).

Von Kossa staining

The mineralization of hPDL cells in the presence of HA and BioCaS was identified by Von Kossa staining. The control and test groups were fixed with 4% PFA for 1 h on 7, 14, and 21 days. The fixed samples were rinsed with deionized water and 2% silver nitrate solution was added to each samples. The samples were then exposed to UV light for 30 min and washed again in deionized water. Calcium deposits were analyzed under the microscope (Nikon TS100, Japan).

Masson's trichrome staining

Masson's trichrome staining was done to evaluate the in vitro collagen deposition of the cultured hPDL cells. The cells were fixed at 7, 14 and 21 days with 4% PFA for 1 h, after thorough washing with PBS. The cells were then washed with deionized water, three times for 5 min each. The cells were treated with freshly prepared Bouin's solution and incubated for 1 h, washed with deionized water, and the nucleus was stained with hematoxylin for 5 min. After washing, the cells were sequentially treated with the component reagents of Masson's trichrome solution (Sigma, Aldrich), following manufacturer's instructions. The stained samples were viewed under the microscope and images were taken.


   Results Top


Isolation and characterization of hPDL cells

The isolation procedure to retrieve hPDL cells from freshly extracted teeth and the explant culture method resulted in cell outgrowth visible from 7 days onwards. Once confluent, these cells were subcultured by normal trypsinization [Figure 1]. The immunoflourescence staining of the first passage cells showed positive expression of the fibroblast marker vimentin, mesenchymal stem cell markers CD90, CD105 and Stro1 and the ligament specific marker Scleraxis. This confirms that periodontal ligament cells exhibiting fibroblast morphology consists of a population of progenitor cells similar to mesenchymal stem cells (MSCs) and also indicates the ligamental nature due to the presence of scleraxis. The presence of the type III intermediate filament protein Vimentin confirms the fibroblast nature of the cells with mesenchymal origin.[16] Mesenchymal stem cells also exhibit fibroblast morphology and were plastic adherent. CD 90 (Thy 1), CD 105 (Endoglin) and Stro 1 are specific cell surface proteins that are accepted MSC markers, the presence of which indicates a population of stem cells within the isolated hPDL cells [Figure 1].
Figure 1: a) hPDL Cell outgrowth from the periodontal tissue fragment kept as explant; b) Expression of intermediate filament marker Vimentin, characteristic of fibroblasts c) Expression of ligament specific marker Scleraxis, characteristic of ligamental nature of periodontal ligament. d, e, f) Expression of mesenchymal stem cell markers CD 90, CD105 and STRO 1

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Cytotoxicity

Cytocompatibility is a necessary screening criterion for biomaterials intended for in vivo use. As a preliminary part of the cell-material interaction studies, cytotoxicity of BioCaS was assessed by direct contact of hPDL cells. The test showed no evidence of cell death or toxicity in the presence of BioCaS cement discs [Figure 2]. The cells maintained their fibroblast morphology, with “Grade 0” scoring (noncytotoxic).
Figure 2: a, b, c) hPDL cells in the presence of BioCaS cement discs at 24 h, 7, and 14, days respectively. d, e, f) hPDL cells in the presence of sintered HA discs at 24 h, 7, and 14 days respectively

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Cytocompatibility

The hPDL cells adhered on BioCaS and HA discs were analyzed by FDA-PI Live-Dead imaging [Figure 3]a and [Figure 3]b. BioCaS showed a majority of viable cells. The actin staining revealed an intact cytoskeleton indicating good cell adhesion and spreading which is remarkably better than with HA [Figure 3]a, [Figure 3]c and [Figure 3]d. SEM images of hPDL Cells on BioCaS and HA discs [Figure 3]b, [Figure 3]e and [Figure 3]f showed lamellipodia and filopodial extensions indicating good cell adhesion and spreading. The cells are seen adherent to the crevices in the BioCaS discs. Some crystalline deposits are also visible.
Figure 3: A. (a and b) 24 h FDA/PI live--dead images of hPDL cells on BioCaS and sintered HA discs, respectively. c, d) Actin cytoskeleton staining of hPDL cells on BioCaS and sintered HA discs, respectively. B. (a and b) SEM images of hPDL Cells on BioCaS and sintered HA discs

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MTT assay

The metabolic activity, analyzed by MTT Assay after 24 h showed similar levels of metabolic activity by hPDL cells (>80%) in the presence of the BioCaS cement discs and sintered HA discs, compared with the cells grown without test materials [Figure 4]. This proved that the biomaterials or their dissolution products did not have any adverse effect on the metabolic activity of the cells.
Figure 4: Percentage metabolic activity of hPDL cells in BioCaS and HA by MTT assay. The hPDL cells showed more than 80% metabolic activity in the presence of the biomaterials

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Osteogenic differentiation of hPDL cells in the presence of BioCaS cement

The hPDL cells cultured in the presence of BioCaS cement discs exhibited mineral deposits similar to the cells cultured in the induction medium, and the results were analyzed histochemically as follows.

Alizarin red staining

The hPDL cells cultured in the presence of BioCaS showed evidence of calcium deposits as early as day 7, when calcium deposits were not present in the positive control. Dense calcific nodules were seen at 14 and 21 days in hPDL cells cultured in the presence of BioCaS [Figure 5].
Figure 5: Alizarin red staining of hPDL cells. a, b, c) The hPDL cells cultured in the presence of BioCaS cement at 7, 14, and 21 days. d, e, f) The hPDL cells cultured in the presence of sintered hydroxyapatite at 7, 14, and 21 days. g, h, i) hPDL cells in osteogenic induction medium at 7, 14, and 21 days (positive control) j, k, l) hPDL cells in regular medium at 7, 14, and 21 days (negative control) The hPDL cells cultured in the presence of BioCaS cement showed early mineralization at 7 days. (a) Mineralization was evident in the form of nodules at 14 and 21 days (b and c respectively) in comparison to the positive control (h and i). The HA group and negative control showed little mineralization (d, e, f and j, k, l), respectively

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Alizarin red assay

The Alizarin red assay results show significant mineralization in the presence of BioCaS cement discs, similar to that of the osteogenic induction group at 21 days [Figure 6].
Figure 6: Alizarin red assay for quantification of mineralization of hPDL cells in the presence of BioCaS and HA. The cells exhibited significantly greater mineralization in the presence of BioCaS than with sintered HA

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Von Kossa staining

The Von Kossa staining showed the presence of mineralized deposits in hPDL cells cultured in the presence of BioCaS cement in regular medium, similar to that of the cells cultured in the induction medium. Furthermore, the cells cultured in the presence of BioCaS showed evidence of early mineralization at 14 days, than the induced group. This is in accordance with the alizarin red values, which also showed evidence of early mineralization [Figure 7]. The hPDL cells cultured on HA did not show the presence of mineralized nodules. This absence of mineralization in HA treated samples is notable, the implications of which are mentioned in the Discussion section.
Figure 7: Von Kossa staining for mineral deposits. a, b, c) The hPDL cells cultured in the presence of BioCaS cement at 7, 14 and 21 days. d, e, f) The hPDL cells cultured in the presence of sintered hydroxyapatite at 7, 14, and 21 days. g, h, i) hPDL cells in osteogenic induction medium at 7, 14 and 21 days (positive control) j, k, l) hPDL cells in regular medium at 7, 14, and 21 days (negative control). Von Kossa staining of hPDL cells showing brownish black deposits, which is evident in the BioCaS group- a, b and c at 7,14, and 21 days, respectively. Mineral deposits in the positive control are evident at 14 and 21 days (h, i). HA group and negative control did not show much mineral deposits

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Masson's trichrome staining

Collagen deposition by the culturing hPDL cells stained by with Masson's Trichrome showed bluish green deposits of collagen in hPDL cells in presence of induction medium and BioCaS cement discs at 14 and 21 days [Figure 8]. The non-induced cells and hPDL cells cultured in the presence of HA did not show evidence of collagen formation, when compared with the cells in induction medium or the cells cultured in the presence of BioCaS. For ideal osteogenic differentiation, collagen deposition and the mineralization of collagen fibers is a necessary parameter.
Figure 8: Masson's Trichrome staining for collagen deposits. a, b, c) The hPDL cells cultured in the presence of BioCaS cement at 7, 14 and 21 days. d, e, f) The hPDL cells cultured in the presence of sintered hydroxyapatite at 7, 14, and 21 days. g, h, i) hPDL cells in osteogenic induction medium at 7, 14 and 21 days (positive control) j, k, l) hPDL cells in regular medium at 7, 14, and 21 days (negative control) Collagen deposits are stained bluish green in the BioCaS group and positive control. (b and h at 14 days; c and i at 21 days. Collagen deposits were absent in HA group and negative control

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   Discussion Top


In the present study, hPDL cells were isolated and cultured successfully and their responses to the in-house developed bioactive materials were tested using an in vitro culture model. Any graft/GTR/GBR material used in periodontal repair will interact primarily with the periodontal ligament cells, and hence hPDL cells were chosen for this study. It has already been reported that MSCs can be isolated from the periodontal tissue and differentiated into osteogenic and cementogenic lineage, under suitable in vitro culture systems.[18] The explant culture method used in the present study gave cell outgrowths from periodontal ligament within 2 weeks. The cells exhibited fibroblast morphology in culture. Primary cells were characterized by the presence of Vimentin, a type III intermediate filament protein present in fibroblasts. The cells were also characterized by the expression of candidate markers of MSC such as CD90, CD105, and Stro1.[19] The hPDL cells also retained their ligamental nature as confirmed by the presence of Scleraxis, a ligament specific marker [20] [Figure 2]. The results of the interactions with material, as depicted in the results, indicate that primary periodontal ligament cells provide an adequate in vitro system to assess the performance of suitable candidate biomaterials for periodontal grafting.[21]

Calcium sulfate (Plaster of Paris) has been in use as a bone filler being a simple and inexpensive cement. It is an established fact that calcium sulfate can induce mineralization in vitro by means of release of calcium ions. The subsequent increase in calcium ions in the extracellular environment is considered to induce osteogenic differentiation and promote mineralization by the cellular calcium sensing mechanisms and the resultant osteogenic gene expressions.[22],[23],[24] The calcium and sulfate ions in biological fluids can enhance the precipitation of calcium phosphates and carbonated hydroxyapatite. Calcium sulfate cement, being porous, can also act as a matrix for the migration and attachment of host cells and provide a lattice for the deposition of amorphous calcium phosphates leading to biomineralization. A transient drop in pH may help in the dissolution of adjacent bone matrix, releasing the growth factors for new bone formation in vivo.[25],[26] Studies mention the use of calcium sulfate as barrier materials along with other autogenous, allogenous, or alloplastic bone graft materials and biologics like PRF.[22],[27] However, the clinical performance has been considered inadequate for bone healing, because of the fast resorption in vivo, before the formation of new bone in the defect.[26]

The BioCaS used in the present study incorporates hydrogen orthophosphate ions which alter the in vivo resorption and bioactivity. The material exhibited good biocompatibility along with successful osteotransduction, the process of bioresorption at the rate of bone formation.[12],[13] The successful in vivo bone healing response of BioCaS cement was inspirational in exploring its use as an alveolar bone graft material for enhancing the regeneration of the periodontium in the present study. The results obtained reassure the cytocompatibility of the material in vitro, with primary hPDL cells.

BioCaS is found to be comparatively more efficient in the osteogenic differentiation of hPDL cells than HA in vitro. This can be attributed to the modification of the material, incorporating hydrogen orthophosphate ions. In addition to the release of calcium ions, the phosphate ions are also released into the extracellular environment during the dissolution of BioCaS cement. The increased concentration of extracellular phosphate ions can induce the differentiation of cells to osteogenic lineage by intracellular mechanisms.[28] Lack of collagen deposits and calcium deposits in the HA group denotes the absence of osteoinductivity of sintered HA, despite being the bone mineral. The reason for the inferior performance may be that the sample was made in dense ceramic form through sintering process. This reduces the dissolution of calcium and phosphate ions, thereby limiting their availability for calcium phosphate formation in presence of hPDL cells.[20]

An experiment design was adopted in the present study for analyzing osteogenic differentiation of hPDL cells in the presence of BioCaS without osteoinduction media. This method provides evidence on the in vitro mineralization potential of BioCaS cement, an observation which can further substantiate the inherent osteogenic potential of the material, uninfluenced by additional osteogenic supplements. The effect of osteogenic inducing agents can be seen in the positive control, to which the BioCaS cement is compared. The presence of calcium, phosphates, and collagen formation by the hPDL cells cultured with BioCaS is an evidence of the osteogenic potential of BioCaS.[29] This study model is a useful tool to evaluate the regeneration potential of similar materials developed for periodontal applications like the new generation GTR/GBR membranes.

HA was used as a test control because it is a known osteoconductive material and is an established biocompatible bone graft material for alveolar bone regeneration. Conventionally, in vitro osteogenic response of HA material with MSCs is found in the presence of inducing agents.[29] In the present study, osteogenic inducing agents were not used in the test groups. The BioCaS group showed evidence of mineralization and the HA group did not. As mentioned above, the increased availability of calcium and phosphate ions from the BioCaS cement may be responsible for the in vitro osteogenic potential observed.

In addition to the osteogenic differentiation, the hPDL cells showed good viability, adhesion and spreading on the BioCaS cement discs. The presence of healthy endogenous progenitor cells, in this case the hPDL cells, and their migration and homing are essential for tissue regeneration.[9],[10] BioCaS can provide a suitable substrate for periodontal ligament cell homing, promoting regeneration. The adherent cells can lay down newly formed collagen between the cementum and alveolar bone. BioCaS also exhibits osteotransductivity, the property of controlled biomaterial resorption at par with the rate of bone formation.[14] This controlled resorption can also help in the insertion of periodontal ligament fibers into the newly formed alveolar bone, promoting healthy regeneration of periodontium. Previous in vivo studies have proven the efficiency of BioCaS as a resorbable bone cement, with good setting properties and adequate working time.[12],[13],[14] Our results reiterate its potential use as an alveolar bone graft material and barrier material for periodontal regeneration.

The drug loading ability of calcium sulfate is useful in local delivery of suitable drugs, especially in cases of localized infections where systemic antibiotics have limited use, due to relatively reduced vascular supply. BioCaS can be loaded with antibiotics for localized drug delivery, which is advantageous in treating localized periodontitis.[23] The osteotransductivity of BioCaS cement can be utilized for alveolar bone regeneration, in association with periodontal regeneration. BioCaS is an ideal “barrier graft” material as it can act as a bone graft for alveolar bone regeneration, and as a resorbable barrier that prevents epithelial migration to aid in periodontal regeneration.


   Conclusions Top


This study explored the osteogenic potential of the new BioCaS bone cement to be used as a graft material for periodontal defect repair. Primary periodontal ligament cells were isolated from freshly extracted human teeth and cultured for the study. The hPDL cells obtained exhibited fibroblast morphology and found to consist of a population of progenitor cells similar to MSCs with ligamental nature. When tested in presence of the cultured hPDL cells, BioCaS cement was seen to promote in vitro mineral deposition without additional supplements. The performance of the new cement was superior to sintered hydroxyapatite ceramics. The cement was found to be cytocompatible as a substrate for cell adhesion and homing, and osteoinductive, the features that can aid in predictable regeneration of periodontium. BioCaS cement is more economical and bioresorbable than sintered HA as bone graft substitute. The results warrant the use of BioCaS cement as a “barrier graft” material for regeneration of periodontal defects. The cell culture model used in this study with cultured hPDL cells proves efficient to evaluate and compare the biomaterials for periodontal grafting.

Acknowledgements

The authors acknowledge with gratitude, the technical help rendered by Mr. Vinod D (Division of Tissue Culture) and Mr. Nishad K. V. (Scanning Electron Microscopy) for conducting this study.

Financial support and sponsorship

The study was conducted with financial assistance from the Dept. of Biotechnology, India.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Dr. Manoj Komath
Division of Bioceramics, Biomedical Technology Wing, Sree Chitra Thirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala - 695 012
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.IJDR_12_18

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