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 Table of Contents  
DPU: INTERDISCIPLINARY CONFERENCE
Year : 2020  |  Volume : 7  |  Issue : 5  |  Page : 91-94

Role of praval bhasma in inducing osteogenic potential in human dental pulp stem cells


1 Department of Oral and Maxillofacial Surgery, Dr. D. Y. Patil Vidyapeeth, Pune, Maharashtra, India
2 Department of Rasashastra and Bhaishajya Kalpana, Dr. D. Y. Patil Vidyapeeth, Pune, Maharashtra, India
3 Director (Research), Dr D Y Patil Vidyapeeth, Pune, Maharashtra, India
4 Dental Consultant, Pune, Maharashtra, India

Date of Web Publication26-Feb-2020

Correspondence Address:
Uday Suresh Londhe
Department of Oral and Maxillofacial Surgery, Dr. D. Y. Patil Vidyapeeth, Pimpri, Pune-18, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jdrr.jdrr_74_19

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  Abstract 


Background: The repair and regeneration of bone is a major issue in the oral and maxillofacial surgery and for the whole human body in general. Bone defects may result from trauma, oncologic surgeries, acute or chronic infections, and congenital malformations. Stem cells are natural and direct source from which stable differentiated cells are generated which lead to tissue formation. Mesenchymal stem cells exhibit the potential to differentiate into a variety of cells such as osteoblasts, myoblasts, chondrocytes, and adipocytes when subjected to various preconditioning agents. Praval Bhasma has never been tested as a preconditioning agent in human dental pulp stem cells (hDPSCs). Materials and Methods: Pulp was extirpated from the teeth of patients who fit in the selection criteria. HDPSCs were then isolated, characterized, and differentiated. Praval Bhasma was used as a preconditioning agent to replace each ingredient in standard osteogenic cocktail and individually to induce osteogenic potential in hDPSCs. Results: Praval Bhasma-induced osteogenic differentiation in hDPSCs when used to replace an ingredient in standard osteogenic cocktail and also when used individually. Conclusion: The use of Praval Bhasma may prove to be a better preconditioning agent for bone formation in hDPSCs.

Keywords: Human dental pulp stem cell, mesenchymal stem cells, Praval Bhasma


How to cite this article:
Londhe US, Dubewar AP, Bhate K, Bhonde R, Shetty L, Kulkarni DG. Role of praval bhasma in inducing osteogenic potential in human dental pulp stem cells. J Dent Res Rev 2020;7, Suppl S2:91-4

How to cite this URL:
Londhe US, Dubewar AP, Bhate K, Bhonde R, Shetty L, Kulkarni DG. Role of praval bhasma in inducing osteogenic potential in human dental pulp stem cells. J Dent Res Rev [serial online] 2020 [cited 2020 Apr 8];7, Suppl S2:91-4. Available from: http://www.jdrr.org/text.asp?2020/7/5/91/278910

Editor: Dr. Pradnya Kakodkar





  Introduction Top


The repair and regeneration of bone is a major issue in the dentofacial orthopedics and for the whole human body in general. Bone defects may result from trauma, oncologic surgeries, acute or chronic infections, and congenital malformations.

The materials used in bone regeneration or in reconstructive surgery must be safe, biocompatible, biodegradable, and should have osteogenic potential. The materials also should facilitate revascularization and be easily incorporated into the recipient site.[1]

Currently, the materials that best meet these requirements are of autologous origin, but autologous bone grafts are limited by donor site pain and morbidity, secondary surgery, bone resorption, osteonecrosis, paraesthesia, cutaneous nerve damage, vascular injury, infection, fracture, and chronic pain.[1]

Allogenic bone and biosynthetic materials are limited by biocompatibility, infection, immune rejection, and graft displacement.[2],[3]

It has only been recently that the dental field has taken a closer look at stem cells and their use not only in promoting more predictable bone grafting but also in the reconstruction of the entire dental tissue. Before this, research on stem cells was being concentrated on the healing of diseased and/or traumatized tissues and organs.

Mesenchymal stem cells (MSCs) exhibit the potential to differentiate into a variety of cells such as osteoblasts, myoblasts, chondrocytes, and adipocytes when subjected to various preconditioning agents.[1]

Osteogenic differentiation protocols using “Standard Osteogenic Cocktail” (dexamethasone [Dex], ascorbic acid [Asc], and β-glycerophosphate [β-Gly]) are frequently used for many experimental approaches, including tissue engineering approaches or simply for the approval of differentiation capabilities of particular cell type.[4],[5],[6],[7]

In osteoblast-like cell cultures, the short-term application of Dex increases bone formation, but the long-term use of Dex decreases bone formation. Gly has very little osteogenic potential. Asc enhances osteoblastic differentiation by increasing collagen accumulation, which results in increased alkaline phosphatase expression in some osteogenic cells.[4] Except for Asc, Dex and gly have little osteogenic potential of their own.[8] Moreover, it requires 3 weeks to get maximum of only 30%–40% osteogenic differentiation from MSCs.

Hence, there is need to search for newer preconditioning agents having better osteodifferentiation potential on human dental pulp stem cells (hDPSCs) in tissue engineering.

Praval Bhasma [Figure 1] has never been tested as a preconditioning agent in hDPSCs.
Figure 1: Raw Prawal

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Praval Bhasma is used in Ayurveda for the treatment of osteoporosis.[9],[10] In human bone, amorphous calcium carbonate is formed as a precursor of the crystalline carbonated apatite/hydroxyapatite.[11] The calcium carbonate (CC) skeleton of marine corals has been reported to be rapidly biodegradable and osteoconductive. The transformation of the CC surface (degradation and new crystal formation) is a prerequisite for osteoblastic apposition and differentiation of osteoprogenitor cells into osteogenic cells, which ultimately leads to bone formation.[3]

The Charak Sanhita Siddhisar, 12/64 states Puranacha punarvamm, i.e.”The ancient medicines can be used for newer inventions”. Hence, in this study, we would like to evaluate the role of Praval Bhasma as an ingredient in “Standard Osteogenic Cocktail” to induce osteogenic potential in hDPSCs with respect to the quality of bone regeneration.


  Materials and Methods Top


This is a prospective, experimental, in-vitro study. The study was carried out in the Regenerative Medicine Laboratory, D Y Patil Dental College, Primpri, Pune, Maharashtra, and the Praval Bhasma was prepared in the D Y Patil College of Ayurveda. A total sample size of 5 teeth which fulfilled the inclusion criteria of noncarious, not infected, and indicated for extraction were collected for the study. The exclusion criteria included the pregnant/lactating women, tobacco in any form, history of any systemic diseases such as hypertension, diabetes, arthritis, bone metabolic disorders, radiation therapy, immunosuppressive therapy, cancer, participants with immunodeficiency virus Types 1 and 2 (HIV-1 and HIV-2), human T-lymphotropic virus Types 1 and 2 (HTLV-1 and HTLV-2), hepatitis A virus, hepatitis B virus, hepatitis B surface antigen, hepatitis C virus, West Nile virus, Epstein–Barr virus, cytomegalovirus, and syphilis and patients unwilling for informed consent. Valid informed written consent was obtained from all the patients.

Before starting the study, permission was obtained from the Institutional Research Board and Institutional Stem Cell Ethics Committee.

Extraction of tooth and removal of pulp

The hDPSCs were obtained from the pulp of freshly extracted healthy teeth [Figure 2].
Figure 2: Sectioning the tooth

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The extracted tooth was immediately placed in a nutritional medium (culture tubes filled with medium (Hank's solution). The specimen was transported on ice to regenerative medicine laboratory for further processing. The tooth was sectioned with tooth cutting discs to retrieve the pulp. [Figure 2] and [Figure 3].
Figure 3: Dental pulp

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Pulp tissue was put in alpha-Minimum Essential Medium (α-MEM). The pulpal tissue was then minced into small fragments and then treated with 0.1% Type IV collagenase + 1% Dispase for 15 min under vigorous shaking, centrifugation at 1500 rpm for 10 min, and supernatant was discarded.

The pellet was then suspended in 5 ml α-MEM and again centrifuged supernatant was removed.

The pellet was seeded in 25 cm2 flask (tissue culture flask) with α-MEM (with glutamine and antibiotics penicillin and streptomycin) +10% fetal bovine serum (FBS) and replaced for 4 days.

Flow cytometry for CD positive and CD negative markers was performed.

The confluent cells were subcultured into two flasks by trypsinization to increase cell number (every 8 days) [Figure 4].
Figure 4: Stem cells in culture medium

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In-vitro cultivation of human dental pulp stem cells

The tissue was rinsed with phosphate-buffered saline and transferred to a conical flask containing a freshly prepared enzyme solution (0.2% dispase and 0.1% Type I collagenase) and placed in an incubator shaker.

The supernatant containing the cells was propagated in α-MEM containing 10% FBS and incubated at 37°C with 5% CO2 [Figure 4].

The culture medium was renewed twice a week. hDPSCs were observed under a phase-contrast microscope.[Figure 5] Cells were subcultured at 80%–85% confluency by trypsinization [Figure 5].
Figure 5: Stem cells under contrast microscope

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Characterization of the cultivated Dental Pulp Stem Cells (DPSCs) was done for stem cell markers.

Preconditioning with Praval Bhasma

The hDPSCs were then preconditioned with Praval Bhasma dissolved in a suitable medium. The cells were then identified using the microscope.


  Results Top


Tissue MSCs differentiated in vitro primarily into the cells of mesenchymal lineage such as bone, cartilage, and adipose tissue [Figure 6].
Figure 6: Differentiation of human dental pulp stem cells

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The in vitro derived MSCs expressed a panel of characteristic surface markers such as Thy-1 (CD90), SH-2/endoglin (CD105), SH-3/4 (CD73), and negative for hematopoietic markers such as CD34 and CD45 HLA-DR [Figure 7].
Figure 7: Surface markers for human dental pulp stem cells

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On preconditioning hDPSc with Praval Bhasma, osteoblastic differentiation took place with bone deposition.

Osteoblastic differentiation was seen in hDPSCs preconditioned with various concentrations of Praval Bhasma. Osteogenic differentiation was seen maximum when Praval Bhasma replaced Dex and also when it was used independently [Figure 8].
Figure 8: Osteoblastic differentiation

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  Discussion Top


The standard osteogenic cocktail consisting of Dex, Asc, and β-gly is frequently used to induce osteogenic differentiation in hDPSCs.[4],[5],[6],[7] The cocktail has its own limitations as each of the ingredient has some side effects.[4] Hence, there existed a need to search for some other ingredient to induce osteogenic differentiation in hDPSCs.

In our study, Praval Bhasma was used to replace each of the ingredients of the standard osteogenic cocktail and also was used individually to study its efficacy to induce osteogenic differentiation in hDPSCs. The result of our study showed that maximum osteogenic differentiation was seen when Praval Bhasma replaced Dex and also when it was used independently. This result may be because Praval bhasma is rich in calcium content, which is alkaline in nature. Praval bhasma contains calcium in the form of Ca2+ form, which is considered to be the most compatible forms of calcium supplementation in the body.[12] The use of Praval Bhasma may prove to be a better preconditioning agent for bone formation in hDPSCs. This approach might be a promising method to shorten the treatment period and improve the quality of bone in the process of distraction osteogenesis, bone defects due to trauma, loss of bone tissue due to tumor resection, especially for patients whose osteogenic potential has been compromised by aging, osteoporosis, etc.

The results of our study are promising and prove that Praval Bhasma can be effectively used to induce osteogenic differentiation in hDPSCs. Further studies about the analysis of time required, amount of differentiation, and amount of Praval Bhasma to be used are necessary to establish it as an standard ingredient in osteogenic cocktail.


  Conclusion Top


Autologous DPSCs can be a new tool for bone tissue engineering. hDPSCs therapy is efficient, exhibits low morbidity of the collection site, and is free from diseases incurred by the transmission of pathogens. The regeneration process is fast and efficient. The use of Praval Bhasma may prove to be a better preconditioning agent for bone formation in hDPSCs.

Acknowledgment

Our mentor and pillar of the research in DPU, Dr. Ramesh Bhonde and Vice Chancellor Dr. M J Pawar were the inspiration and support behind it. Dr. D. Gopalakrishnan, Dean, Dr. D. Y. Patil Dental College and Hospital, Pimpri, Pune18 and Prof. Dr. Supriya Kheur, HOD, Department of Oral Pathology supported and guided at each and every step. Prof. Mr. Avinash Kharat and Mr. Vikrant Patil, Regenerative Medicine Laboratory, Dr. D. Y. Patil Dental College, Pimpri, Pune 18 supported in this research work.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Belmonte GC, Holgado LA, de Labio RW, Rosa PS, Segato R, Fukasawa JT, et al. The Potential of Human Dental Pulp Stem Cell from Deciduous Teeth in Bone Regeneration: An Experimental Study in Rabbits. Int J Oral Dent Health 2016;2:032.  Back to cited text no. 1
    
2.
Zhao D, Wang Y, Han D. Periosteal distraction osteogenesis: An effective method for bone regeneration. Biomed Res Int 2016; Article ID 2075317.  Back to cited text no. 2
    
3.
Ohgushi H, Okumura M, Yoshikawa T, Inoue K, Senpuku N, Tamai S, et al. Bone formation process in porous calcium carbonate and hydroxyapatite. J Biomed Mater Res 1992;26:885-95.  Back to cited text no. 3
    
4.
Langenbach F, Handschel J. Effects of dexamethasone, ascorbic acid and β-glycerophosphate on the osteogenic differentiation of stem cells in vitro. Stem Cell Res Ther 2013;4:117.  Back to cited text no. 4
    
5.
Castiglioni S, Romeo V, Locatelli L, Cazzaniga A, Maier JAM. TRPM7 and MagT1 in the osteogenic differentiation of human mesenchymal stem cells in vitro. Sci Rep 2018;8:16195.  Back to cited text no. 5
    
6.
Samsonraj RM, Dudakovic A, Manzar B, Sen B, Dietz AB, Cool SM, et al. Osteogenic stimulation of human adipose-derived mesenchymal stem cells using a fungal metabolite that suppresses the polycomb group protein EZH2. Stem Cells Transl Med 2018;7:197-209.  Back to cited text no. 6
    
7.
Pasini A, Lovecchio J, Ferretti G, Giordano E. Medium perfusion flow improves osteogenic commitment of human stromal cells. Stem Cells Int 2019; Article ID 1304194.  Back to cited text no. 7
    
8.
Park JB. The effects of dexamethasone, ascorbic acid, and β-glycerophosphate on osteoblastic differentiation by regulating estrogen receptor and osteopontin expression. J Surg Res 2012;173:99-104.  Back to cited text no. 8
    
9.
Lata S, Biradar RS. Physio-chemical analysis of Praval Bhasam – Prepared by using Praval Mool as raw material. Int J Ayurvedic Herbal Med 2015;5:1954-63.  Back to cited text no. 9
    
10.
Nageswar Rao V, Dixit SK. Standardisation of Pravala Bhasma. Anc Sci Life 1998;17:203-6.  Back to cited text no. 10
    
11.
Tolba E, Müller WE, Abd El-Hady BM, Neufurth M, Wurm F, Wang S, et al. High biocompatibility and improved osteogenic potential of amorphous calcium carbonate/ vaterite. J Mater Chem B 2016;4:376-86.  Back to cited text no. 11
    
12.
Pal SK. The ayurvedic bhasma: The ancient science of nanomedicine. Recent Pat Nanomed 2015;5:12-8.  Back to cited text no. 12
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]



 

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