Human dental pulp mesenchymal stem cells isolation and osteoblast differentiation

Transcription

1 ORIGINAL ARTICLE Human dental pulp mesenchymal stem cells isolation and osteoblast differentiation Moustafa Alkhalil 1, Amer Smajilagić 1, Amira Redžić 2 1 Department for Oral and Cranio-Maxillo-Facial Surgery, Hamad Medical Corporation, Doha, Qatar, Weill Cornell Medical College, Doha, Qatar 2 Institute for Biology and Human Genetic, School of Medicine University of Sarajevo, Sarajevo, Bosnia and Herzegovina ABSTRACT Aim This study was focused on the isolation and characterization of mesenchymal stem cells (MSCs) from human dental pulp (DPSC). Corresponding author: Amer Smajilagić Department for Oral and Cranio-Maxillofacial Surgery, Hamad Medical Corporation P. B. 3050, Doha, Qatar Phone: ; Fax: ; smajilagica@gmail.com Original submission: 10 September 2014; Revised submission: 22 September 2014; Accepted: 29 October Methods The study was performed in the Department for Oral and Cranio-Maxillo- Facial Surgey Hamad Medical Corporation, Doha, Qatar and Weill Cornell Medical Colleague Doha, Qatar, in period Dental pulp was extracted from premolars and third molars of 19 healthy patients. The pulp was digested in a solution of 3 mg/ml collagenase type I and 4 mg/ml dispase for 1 hour at 37C. After filtration, cells were cultured in Dulbecco s Modified Eagle Medium (DMEM Low Glucoses) with 20% Fetal Bovine Serum (FBS), 2mM L-glutamine and antibiotics (100 U/ ml penicillin, 100 ug/ml streptomycin) at 37 C under 5% CO2. Cultures were treated with osteoinductive medium for differentiation MSC in to the osteoblast cell line. Staining with Alizarin red were used for the detection of the osteoblast production and calcification new formed tissue. Results On the total of three out of 19 patients it was possible to isolate DPMSCs after 2 to 3 weeks: in one patient it was not possible to expand MSCs because of infection, and in other two patients positive Alizarin red staining reaction showed osteogenic differentiation capability and strong mineralization in vitro. Conclusion The main advantage of using DPSC is absence of morbidity. MSCs could be isolated noninvasively from teeth, routinely extracted in the clinic and discarded as medical waste. Standardization of clinical and laboratory protocols for DPMSCs isolation and team work coordination could lead to significantly improved result. Key words: regenerative medicine, bone, tissue engineering, osteoblast Med Glas (Zenica) 2015; 12(1):

2 Medicinski Glasnik, Volume 12, Number 1, February 2015 INTRODUCTION Tissue engineering is an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ. Those involved in regenerative medicine place more emphasis on the use of Stem Cells to produce tissues (1). Stem cells have two main properties: pluripotency, e.g. formation of all ectoderm, endoderm and mesoderm cells layers, and self renewal (2,3). Depending on the origin there are embryonic stem cells and somatic stem cells which include mesenchymal stem cells, hematopoietic stem cells and neural stem cells. Mesenchymal stem cells (MSCs) are able to differentiate into the different cell types such as osteoblast, chondroblast myoblast, adipoblast and even beta- pancreatic cells (4). These cells could be isolated from the variety of tissues and expanded in vitro. Such produced MSC may have the use as valuable tools for regenerative medicine (5,6). Mesenchymal stem cells of human origin were isolated and have been used for the preclinical studies and clinical trials (7,8). The most common source is bone marrow (9-11), fat tissue (12,13) and umbilical cord blood (14-16). However, isolation of the MSCs from those sources is performed by aspiration and is often an invasive and painful procedure for donors, associated with morbidity. Umbilical cord cells are only available immediately after the delivery so they have limited availability. Because of those disadvantages, identification of other sources for MSCs isolations is of great importance (17). This study is focused on the isolation and characterization of MSCs from human dental pulp (DPSC) (18). An important issue is presented indicating that there is a significant diversion in the MSCs isolation protocols from different tissues (19), which makes it difficult to compare results between laboratories and verify success of clinical trials. The aim of this study was isolation and cultivation of MSCs from the human dental pulp as a future model for regenerative medicine application with the intention of establishing unique and effective clinical and laboratory protocol, characterization and differentiation in the osteoblast cells phenotype. MATERIALS AND METHODS For isolation of human dental pulp mesenchymal stem cells (DPMSCs), the extracted teeth of the total of 19 patients treated at the Department for Oral and Cranio-Maxillofacial Surgery, Hamad Medical Corporation, Doha, Qatar were used. In collaboration with the Laboratory for Stem Cells Research Weill Cornel Medical College, Doha, Qatar, the study was performed in the period All patients were healthy based on documented evidence found in their medical files. All patients or their legal representatives signed an informed consent form, following the guidelines approved by the Ethic Committee of the Medical Research Center, Hamad Medical Corporation, Doha, Qatar. All removed teeth used in the study were intact and medically indicated for the extraction, either for orthodontic reasons or causing health problems. Teeth were removed using local anesthesia with 2% xylocain/adrenaline, under standard conditions, and after the removal they were cleaned with 70% alcohol swab topically. Extracted teeth were placed into the sterile plastic tubes, and tubes were put into ice, stored between 0-4 C, for 6-18 hours overnight. This time delay was necessary whilst waiting for transportation to the stem cell laboratory and isolation procedures. All the teeth had fully developed roots and using Luers forceps, in the laboratory the roots were separated from the crown. After the separation an extirpation needle was used to remove dental pulp. After extirpation pulp tissues were digested in a solution contest from 4 ml of DMEM/low Glc, 100U/mL penicillin, 100 ug/ml streptomycin, and 3 mg/ml enzymes collagenase type I and 4 mg/ml dispasae for 60 minutes et 37 C. Then, cells were digested in the media and centrifuged for 10 min at 1200 rpm. The pellet was washed twice in PBS, resuspended in to the 6 ml cell culture medium and placed in T25 flasks. The flasks were incubated in 5% CO 2 at 37 C. The medium was changed twice weekly. The cells cultivate medium were contest of: DMEM/Low Glc with 20% fetal bovine serum (FBS) 2 mm L-glutamine, 100U/mL penicillin and 100 ug/ ml streptomycin. When the cells in 24 well plate reached 50-60% confluency, the medium in each flask was replaced using fresh osteogenic differentiation media contest: ascorbic 28

3 Alkhalil et al. h-mscs dental pulp isolation acid 50 ug/ml, b-glycerol phosphate 10 mm, Dexamethason 10-7 in DMEM/low Glu and 10% FBS, 2mM L-glutamine, 100U/mL penicillin and 100ug/mL streptomycin. Osteogenic medium was changed twice weekly, and after 2 weeks the cells were differentiated and stained with 2% Alizarin, for the detection of osteoblast production and calcification. The Alizarin method for the staining was performed according the procedure: washing the cells 2x with PBS, 10 min with 70% ice cold ehanol, washing with distilled H 2 O, application 10 min Alizarin red at RT, 5 times washing with distilled H 2 O, add PBS and after 15 minute checking under a microscope to see the calcification. RESULTS Dental pulps were extirpated from the extracted teeth, respectively for each individual patient from the total of 19 patients included in study (6 males, 13 females), for the purpose of isolation and cultivation of MSCs. In 16 of them permanent premolars teeth were extracted, according to orthodontist indication in therapeutic purpose, and in the other three patients permanent molars were extracted due to health reasons. The average age of the patients was 13 years (range 9-22 years). From the total of 19 patients, it was possible to detect and isolate MSCs in three of them. The MSCs were detected by visualization colonies on electronic microscope and their specific spherical like cluster shape (Figure 1). The time between extirpation pulp tissue and DPMSCs isolation was variable. In one patient time the duration between isolation and detection of MSCs was 7 days, on another it took 13 days, and on the third 24 days. Two patients were females and one patient was male (average age of 13 years). After visualization, colonies started to grow rapidly in the cell media. After isolation in one patient, an infection appeared and destroyed all the cells. The other two patients (one male, one female) dental pulps were extirpated from the extracted premolars, and showed DPMSCs isolation after 2 weeks, colonies were expanded and when they reached confluences 50-60% they were exposed to the osteogenic differentiation media for next two weeks (Figure 2). A) B) Figure 2. A) Expanded stem cells in culture, single pulp extracted premolar tooth, day 24 after isolation on male patient; B) Expanded stem cells in culture, single pulp extracted premolar tooth, day 30 after isolation on female patient. On both isolated cultures, cells are adherent, elongated in spindle shape with thin expansion (phase contrast microscopy) (Mehteb M, 2010) Figure 1. Colonies of the aggregated stem cells on the 13 th day of culture in a 13-year old male patient, isolated from single dental pulp of the removed premolar tooth. Cells were adherent and formed aggregates. In total 7 colonies of stem cells were detected (Mehteb M, 2010) Two weeks after the exposure to osteoinductive factors, Alizarin red staining of DPMSCs culture demonstrated positive reaction. Long term growth cultures of DPMSCs (between 4-6 weeks) 29

4 Medicinski Glasnik, Volume 12, Number 1, February 2015 in vitro, demonstrated the capacity to form Alizarin red-positive mineralization nodules with high levels of calcium (Figure 3). Figure 3. Adherent layers of cultured DPMSCs are shown (with Alizarin Red staining as a measure of calcium accumulation after induction with Ascorbic acid 50ug/ml, b-glycerol phosphate 10mM, Dexamethason 10-7 in DMEM/low Glu and 10% FBS, 2mM L-glutamine), capacity to form Alizarin Red-positive condensed nodules with high levels of calcium (Mehteb M, 2010) DISCUSSION Human tissues have different potential for regenerative properties. It appears that stem cells and their biology may play an integral role in that regenerative process. Multipotent mesenchymal stem cells (MSCs) were first discovered by Friedenstein 1976 (9), who isolated them from bone marrow. Based on this protocol, MSCs from other tissues were searched for isolation and cultivation. Several loci within human body were isolated as a source for the MSCs isolation: bone marrow, cartilage tissue, fat tissue, and umbilical cord blood (11, 13-15, 20). However, accessibility can be limiting. Mesenchumal stem cells from dental tissue were isolated by Gronthoss 2000 (18) from the pulp tissue. Those cells showed capability to differentiate into the osteoblasts, odontoblasts, adipocytes, and neural cells (21). The results of this study have shown ability of ex vivo expanded human DPMSCs taken from permanent teeth to differentiate into osteoblasts and produce in vitro a mineralized bone like tissue, detected by using a specific calcium deposit staining Alizarin red, similar to some of the previous research (18,19,21). The aim of this study was to isolate (from each individual) DPMSCs as autologous cells model, potential clinical tools which can be used as alternative to conventional medicine, free of any immune response and no ethical dilemma that we have in embriogenic stem cells research. For example, as supplement for bone grafting in conventional surgery, or for use in any clinical situations where the regenerative process is damaged (22). In systematic diseases, such as osteoporosis, therapies are mostly oriented towards suppression osteoclast function but no one or very few are oriented towards osteoblast proliferation (23). Comparing with isolation from other sources of MSCs, DPMSCs are easily accessible tissue resource (24). Most important advantage of this MSCs source is absence of morbidity and no need for additional surgical procedures. Harvesting process of donor tissue is done when a patient comes to have a tooth removed for health or therapeutic reasons, and instead of throwing tooth in the garbage, it should be placed in the process of MSCs isolation and cultivation. Such isolated DPMSCs could be also cryo-preserved and stored for future treatment of that person if required (25). Comparing with DPMSCs, an umbilical cord blood and amniotic fluid are limited MSCs sources, as they are available only after delivery (25) Dental pulp as MSCs source offers multiple opportunities for their isolation throughout life. Some research indicates that the DPMSCs are detectable in humans up to the age of 30 years, which is in accordance with results of this study where the average age of successful isolations was 13 years (26-28). Some investigations show that stem cells can be obtained from the dental pulps of subjects between 30 and 45 years of age using specific antibodies for stromal stem cells (29). Our results also indicate that h-dpm- SCs possess high proliferation ability and great potential for isolation of large numbers of cells, suggesting that they are a useful source for stem cell based therapy. In conclusion, individual autologous stem cells from dental pulp were isolated in only three out of 19 patients, and in two of them osteoblast cells line and calcification tissue were produced. Main disadvantages of this study include the low rate of the DPMSCs isolation and a long time period between pulp tissue isolation and DPMSCs visualization and cultivation. Our findings suggest the main reason for such results was the long time between tooth extraction and 30

5 Alkhalil et al. h-mscs dental pulp isolation pulp tissue extirpation for DPMSCs isolation. Probably, the overnight delay of more than 10 hours could lead to the MSCs and other cells dying. Even that result of 15% successful isolation in such conditions indicates that dental tissue is a great potential reservoir of stem cells, with a high rate of survival. Improvement in the standardization of clinical and laboratory protocols, and better coordination and team work could significantly improve this process. FUNDING This research was supported by the Medical Research Office, Hamad Medical Corporation, Doha, Qatar (Project Project No: 8121/08 Mesenchymal Stem Cells Isolations and Cultivation as Experimental Bone Tissue Engineering System ) and Qatar, Doha, Weill Cornel Medical School, Laboratory for Stem Cells Research and Senior Researcher Mehteb Meleki. TRANSPARENCY DECLARATIONS Competing interest: none to declare. REFERENCES 1. Arthur A, Zannetino A, Gronthos S. The Therapeutic applications of multipotential m esenchymal/stromal stem cells in skeletal tissue repair. J Cell Physiol 2009; 218: Kassem M. Mesenchymal stem cells: biological characteristics and potential clinical applications. Cloning Stem Cells 2004; 6: Abdallah BM, Kassem M. Human mesenchymal stem cells: from basic biology to clinical applications. Gene Ther 2008; 15: Baksh D, Song L, Tuan RS. Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy. J Cell Mol Med 2004; 8: Graziano A, d Aquino R, Laino G. Papaccio G. Dental pulp stem cells. A promising tool for bone regeneration. Stem Cell Rev 2008; 4: Iohara K, Zheng L, Wake H, Ito M, Nabekura J, Wakita H, Iohara K, Zheng L, Wake H, Ito M, Nabekura J, Wakita H, Nakamura H, Into T, Matsushita K, Nakashima M. A novel stem cell source for vasculogenesis in ischemia: subfraction of side population cells from dental pulp. Stem Cells 2008; 26: Yamada Y, Ueda M, Hibi H, Nagasaka T. Translational research for injectable tissue- engineered bone regeneration using mesenchymal stem cells and platelet-rich plasma-from basic research to clinical case study. Cell Transplant 2004; 13: Yamada Y, Nakamura S, Ito K, Kohgo T, Hibi H, Nagasaka T. Injectable tissue-engineered bone using autogenous bone marrow-derived stromal cells for maxillary sinus augmentation: clinical application report from a 2-6-year follow-up. Tissue Eng Part A 2008; 14: Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol 1976; 4: Caplan AI. Mesenchymal stem cells. J Orthop Res 1991; 9: Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997; 276: Mizuno H, Zuk PA, Zhu M, Lorenz HP, Benhaim P, Hedrick MH. Myogenic differentiation by human processed lipoaspirate cells. Plast Reconstr Surg 2002; 109: Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, Alfonso ZC, Fraser JK, Benhaim P, Hedrick MH. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 2002; 13: Romanov YA, Svintsitskaya VA, Smirnov VN. Searching for alternative sources of postnatal human mesenchymal stem cells: candidate MSC-like cells from umbilical cord. Stem Cells 2003; 21: Mareschi K, Biasin E, Piacibello W, Aglietta M, Madon E, Fagioli F. Isolation of human mesenchymal stem cells: bone marrow versus umbilical cord blood. Haematologica 2001; 86: In t Anker PS, Scherjon SA, Kleijburg-van der Keur C, de Groot-Swings G Claas FH, Fibbe WE. Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta. Stem Cells 2004; 22: Huang GT, Gronthos S, Shi S. Mesenchymal Stem Cells Derived from Dental Tissues vs. Those from Other Sources: Their Biology and Role in Regenerative Medicine. J Dent Res 2009; 88: Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci USA 2000; 97: Gronthos S, Zannettino ACW. A method to isolate and purify human bone marrow stromal stem cells. In: Prockop DJ, Bunnell BA, Phinney DG, editors. Mesenchymal stem cells: methods and protocols. Totowa, NY: Humana Press, 2008; Young HE, Steele TA, Bray RA. Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal, adult, and geriatric donors. Anat Rec 2001; 264: R d Aquino R, Graziano A, Sampaolesi M, Lainol G, Pirozzi G, De Rosa, R d Aquino R, Graziano A, Sampaolesi M, Lainol G, Pirozzi G, De Rosa, Papaccio G. Human postnatal dental pulp cells co-differentiate into osteoblasts and endotheliocytes: a pivotal synergy leading to adult bone tissue formation. Cell Death Differ 2007; 14: Graziano A, d Aquino R, Laino G, Papaccio G. Dental pulp stem cells: a promising tool for bone regeneration. Stem Cell Rev 2008; 4:65. 31

6 Medicinski Glasnik, Volume 12, Number 1, February Undale AH, Westendorf JJ, Yaszemski MJ, Khosla S. Mesenchymal stem cells for bone repair and metabolic bone diseases. Mayo Clin Proc 2009; 84: Nakamura S, Yamada Y, Katagiri W, Sugito T, Ito K, Ueda M. Stem cell proliferation pathways comparison between human exfoliated deciduous teeth and dental pulp stem cells by gene expression profile from promising dental pulp. J Endod 2009; 35: Papaccio G, Graziano A, d Aquino R, Graziano MF, Pirozzi G, Menditti D, De Rosa A, Carinci F, Laino G Long-term cryopreservation of dental pulp stem cells (SBP- DPSCs) and their differentiated osteoblasts: a cell source for tissue repair. J Cell Physiol 2006; 208: Suchaneka J, Soukupb T, Visekb B, Ivancakova R, Kucerovac L, Mokryb J. Dental Pulp Stem Cells and their characterization. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2009; 53: Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A. Stem cell properties of human dental pulp stem cells. J Dent Res 2002; 81: Batouli S, Miura M, Brahim J, Tsutsui TW, Fisher LW, Gronthos Set, Batouli S, Miura M, Brahim J, Tsutsui TW, Fisher LW, Gronthos SRobey PG, Shi S. Comparison of stem-cell-mediated osteogenesis and dentinogenesis. J Dent Res 2003; 82: Laino G, D Aquino R, Graziano A, Lanza V, Carinci F, Naro F, Pirozzi G, Papaccio G. A new population of human adult dental pulp stem cells: a useful source of living autologous fibrous bone tissue. J Bone Miner Res 2005; 20: