Research Article
Volume 1 Issue 1 - 2014
The Osteoinductive Properties And Influence of “Emdogain” (Straumann) on Colony-Forming Properties of Human Osteogenous Progenitor Bone Marrow Cells Ex vivo
Biloklytska G F1, Panchenko L M2 and Braun I E1*
1Department of conservative dentistry, National medical academy of postgraduate education named after P L Shupyk, Ukraine
2Institute of traumatology and orthopedics of National Academy of Medical Sciences of Ukraine, Ukraine
*Corresponding Author: Braun Iuliia, Department of conservative dentistry, National Medical Academy of Post-Graduate Education named after P L Shupyk, Dental Scientific Medical Center, Kyiv, Ukraine.
Received: December 23, 2014; Published: December 31, 2014
Citation: Biloklytska G F., et al. “The Osteoinductive Properties And Influence of “Emdogain” (Straumann) on Colony-Forming Properties of Human Osteogenous Progenitor Bone Marrow Cells Ex vivo”. EC Dental Science 1.1 (2014): 25-32.
Abstract
Generalized periodontitis (GP) is widespread disease among working category of patients that is very actual for Ukraine where periodontology still is not recognized specialty. Modern data demonstrate presence of connection and influence of GP on development of chronic diseases that is very important point for general health care of citizens, especially of Ukraine. According to Biloklytska G., an effective treatment of GP for citizens of Ukraine is among priorities, where statistics of disease prevalence shows very high percent of patient`s involvement. Growing tendency among our patients revealed fact of great want of patients to leave natural tooth on place instead of extraction. This fact enforces practicing dentists to search for more favor and biology based treatment techniques of GP. Especially it is important during usage of additional materials and medications for periodontal regeneration. This means that all used modern materials should fulfill all the demands, especially when we deal with regenerative periodontology, when the periodontal regeneration highly expected. Usage of enamel matrix proteins in periodontology is still very perspective promoting periodontal regeneration, influencing on different parts of periodontal wound regeneration. Also the regeneration of bone tissue as component of periodontal complex can be enhanced due to biological mechanisms support. In the review the preliminary data about details of occurrence of osteoinductive properties of “Emdogain” (Straumann) and the influence on colony forming properties of osteongenous bone marrow cells will be showed. The preliminary results will be showed and discussed.
Keywords: Generalized periodontitis; Periodontal regeneration; Enamel matrix proteins; Emdogain; amelogenin; Osteogenous progenitor human bone marrow cells; Colony forming activity; Osteinduction
Abbreviations: GP: Generalized Periodontitis; PD: Periodontal Disease; PDL: Periodontal Ligament; EMPs: Enamel Matrix Proteins; APP: Acute Phase Proteins; HERS: Hertwig`s Epithelial Root Sheath; ALP: Alkaline Phosphatase; OC: Osteocalcin; TGF-β: Transforming Growth Factor β; CTGF: Connective Tissue Growth Factor; VEGF: Vascular Endothelial Growth Factor; HMVECs: Human Microvascular Endothelial Cells; BMP: Bone Morphogenetic Protein; BSP: Bone Sialoprotein; OPG: Osteoprotegerin; CFFU: Colony-Forming Fibroblast Units
Introduction
Periodontal disease (PD) involves the chronic and progressive destruction of the tooth supporting tissues, mainly the periodontal ligament (PDL), root cement and alveolar bone, whose regeneration is most complicated and under present scientific interest [1-5]. This fact can be explained by all collected data about complex structure and functional details of these data [1-8]. However tooth loss as the most widespread consequence that means exact data about all regenerative mechanisms are absent [3-5,9,10]. A major discovery was the finding that periodontal tissue regeneration could be achieved by application of acidic extract of enamel matrix proteins (EMPs), and later- by the FDA approved medical device termed Emdogain [3-5,11-14]. It is well also known that PD cause chronic and progressive destruction of the tooth supporting tissues, which attaches the tooth to the alveolar bone of the jaw, leading to progressive bone resorption process [3-5,13,15]. But the complete periodontal regeneration is still under plural investigations, profound investigations are not able to supply understanding of all cell-to-cell regenerative mechanisms [1,2,5,7,14-18]. Also regeneration of root cement is among main scientific interest, having different cement types, cells whose function and genesis are still unknown [1,2,5,7,14,18].
And regeneration of the periodontium is a major goal in the treatment of teeth affected by periodontitis [1,2,5,7,14,17,19]. It was demonstrated, in the periodontal regeneration model, migration of cells from the adjacent unwounded PDL into the wounded area [16], bone marrow stromal cells also have the potential to regenerate the periodontium [20]. It was demonstrated that amelogenin stimulates cascade of events due to remodeling and regeneration of periodontal tissues [3,4,7,14]. Another data showed the expression of amelogenin mRNA isoform-M180 in macrophages, suggesting the endogenous expression of amelogenin in osteoclasts [21]. Also authors suggested that during alveolar bone regeneration after induced inflammation, amelogenin might act by increasing bone formation over resorption process. It may function in the fine balance between bone formation by osteoblasts and bone resorption by the osteoclasts, possibly through the RANKL/RANK mediated pathway [22,23]. Amelogenin expression is highest in bone cells adjacent to PDL and in the periosteal cells, as compared to bone cells distal to the PDL and the periosteum [3,4]. This can appear because the bone adjacent to the PDL absorbs all the mastication forces and bone turnover, remodeling are much higher in this area, as compared to bone remote to the PDL region [3-5]. Periosteum is the main site of bone growth and remodeling, and it contains mainly progenitor cells [5]. The findings from animal study on dogs suggest that in the normal uninjured animal, amelogenin expression is increased at sites of high bone activity and remodeling [3]. A similar phenomenon was seen in the regenerated alveolar dog bone; amelogenin was highly expressed in endosteal cells, osteoblasts, and cells surrounding blood vessels in the regenerated area, and to a much lesser extent in bone cells distal to the regenerated area [4]. Authors detected amelogenin expression both in osteoclasts and in osteoblasts lining the alveolar bone trabeculae [3,4].
Pericytes whose are involved in regulating of angiogenesis, differentiate along several lineages including osteogenic and chondrogenic phenotypes. The finding that amelogenin might promote angiogenesis in the regenerating tissues explained in in vitro and in vivo findings demonstrating chemotaxis of endothelial cells and induction of blood vessel formation by EMPs [24,25]. Chronic periodontal disease is similar to systemic inflammation characterized by the mediators such as acute phase proteins, inflammatory cytokines as IL-6 and coagulation factors as a fibrinogen [19,17]. PD could be the case of the generalized acute phase response [9,10]. Among others also changes in the level of numerous serum proteins occur these proteins are therefore called acute phase proteins (APP) [9]. Several biological activities of APPs were described e.g., enhancement of nonspecific response complement activation, involvement in blood clotting and fibrinolytic activities defence against iron lost [9,10]. Some APPs participate in limiting the inflammatory reaction and in clearance of its products, the facilitate phagocytosis; participate in repair processes by influencing fibroblast activity and reorganization of new developed connective tissue healing wound. They may also affect activity of the immune cells [8-10,16]. Oxidative stress also is postulated to be increased in patients with periodontitis and oral mucosa diseases. It is suggested that oxidative cell injury caused by reactive oxygen species contributes to the development of inflammatory changes of oral mucosa [9]. Knowing the fact that Hertwig`s epithelial root sheath (HERS) cells have synthetic activity, they also secrete molecules into extracellular milieu [1,2]. While some studies showed EMP expression on the developing root surface [1,2,7,14], others have claimed that HERS` cells produce cementum proteins [1,2]. Based on findings of the occasional expression of EMPs along the forming root, the idea developed that EMPs plat pivotal role in the differentiation of progenitor cells into cementoblasts that specifically produce acellular extrinsic fibre cementum [7,14]. Systemic review and meta-analysis concluded that periodontal therapy does not result in a significant reduction of serum acute phase proteins levels [26], as well as alfa 1-antichymotrypsin [6], and alfa 1-acid glycoprotein [6]. Emdogain is an acidic extract of extracellular enamel matrix, and induces a heterogeneous mixture of mainly hydrophobic polypeptides encoded by several genes [7,14,27]. It was not clear however, which of the EMPs induce the regeneration, and what molecular mechanisms are involved [27]. It was not clear however, which of the EMPs induce the regeneration, and what molecular mechanisms are involved [27]. For decades amelogenin was thought to be exclusively an enamel (epithelial origin) protein. However, in more recent years, amelogenin has also been detected in dentin matrix [7,14,28], odontoblasts [29], in remnants of Hertwig`s root sheath and in PDL cells [30,31], in long bone cells some of which are multi-potent stem cells [3], osteocytes, osteoblasts, osteoclasts, cartilage chondrocytes, in growth plate cells [4]. The relatively large number of amelogenin alternatively spliced mRNA translated polypeptides and the fact that amelogenin is expressed in different tissues (calcifying and soft tissues) and of different embryonic origin, possibly reflect different functions of amelogenin [4]. Application of amelogenin induces local neovascularization to support the developing tissues, and possibly might change the fine balance between osteoclast and osteoblast activity, favoring regeneration over the natural process of degeneration after induction of chronic periodontitis in experiment. This degeneration process is characterized by connective tissue and epithelial proliferation into injured area, an event thought to prevent proper regeneration of the periodontal tissues [4]. The bone matrix is known to contain BMPs, highly potent growth/differentiation factors that induce the differentiation of progenitor cells into osteoblasts. However, the dentin matrix appears to have a higher osteoinductive capacity [1,2,32,33]. in vitro and in vivo studies showed the chondrogenic and osteogenic activities of recombinant amelogenin polypeptides [32,33]. These findings are in line with those obtained with enamel matrix and amelogenin [34,35]. However another authors showed that EMD failed to enchance proliferation of stromal osteoblastic cells obtained from the bone marrow of young adult male rats [12]. In contrast, treatment with EMD of human osteoblastic cells (SaM-1) from one patient [36] and of rat femoral bone marrow stroma [37], mouse preosteoblast cell line [38], significantly stimulated cell proliferation. However, when alveolar bone cells were used, no effect of EMD on cell proliferation was observed, but the bone cells showed the greatest attachment response to EMD [39]. In contrast, it was showed that growth of human mandibular osteoblasts from one patient was significantly increased by EMD [40]. In heterogeneous cell population from rat bone marrow, EMD had no significant effect on cell proliferation, alkaline phosphatase (ALP) activity, and mRNA expression of type I collagen, osteocalcin (OC) [18]. Using an organoid culture system with human primary osteoblasts it was observed a significant increase in cell proliferation [41]. It was observed that EMD promotes motility of different osteoblastic cell lines better than the control groups, whereas the proliferation rates depended on the cell type [42]. In another study where a chondrogenic cell line, EMD markedly increased cell proliferation [43]. It was showed that EMD stimulated proliferation of human bone marrow stromal cells in a dose-dependent manner [11]. EMD treatment of human osteoblastic cells (Saos-2) increased cell proliferation [44]. Using human bone marrow stromal cells, it was showed that EMD down regulated type I collagen synthesis and ALP activity, whereas the decrease in OC synthesis was not statistically significant. in vitro mineralization was reduced in EMD-treat cells [11]. Also authors showed that EMD up-regulated the release of TGF-β1 from primary rat osteoblasts into the culture medium [45]. Also when human osteoblastic cells (Saos-2) were treated with EMD, a dose-dependent increase in the expression of connective tissue growth factor (CTGF), a mediator of TGF-β1 was observed [44]. EMD-induced CTGF expression and in vitro mineralization were significantly reduced in the presence of TGF-β inhibitor and treatment with anti-CTGF antibody, respectively. Using the bone wound-healing model in rat femurs [46] and in rat parietal bone [47], authors noted a significantly higher bone volume fraction of newly formed bone volume trabeculae 7 days after injury in the EMD group compared with the control [48]. Also authors suggested that effects of EMD also depend on the local osseous environment. It was showed that exposure of different cell types like: PDL cells, gingival fibroblasts, osteosarcoma cell line on EMD, resulted in enhanced wound-fill rates [48,49]. The early healing stages showed more effect on PDL than of gingival fibroblasts. The effects of EMD on microvascular endothelial cells (HMVECs) were investigated and it was suggested that low concentrations resulted in significant stimulation of HMVEC proliferation, and HMVEC chemotaxis when PDL cells were present [50]. All doses tested increased angiogenesis. HMVECs in combination with EMD stimulated a 750% increase in PDL cell migration compared with controls. Also it was determined at almost 400% increase in VEGF concentration by ALP-positive PDL cells and a significant increase in TGF-β production in both ALP-positive and ALP-negative PDL cells in EMD-stimulated conditioned media [50]. Also authors demonstrated that in human PDL cells EMD down-regulates the expression of genes involved in early inflammatory events of wound healing, whereas genes encoding growth and repair-promoting molecules were up-regulated [51]. The increased expression of BMP-2, BMP-7, BSP, cementum attachment protein-23 (CP-23) was noted and two putative cementum markers [52]. EMD significantly stimulated total protein synthesis by human PDL fibroblasts [49]. EMD exposure to human PDL fibroblasts resulted in significantly enhanced osteocalcin (OC) and osteoprotegerin (OPG) levels [8].
Even if it was showed existence of not significant statistical difference in between groups where EMD were applied or not [9,10], better tendency for healing in EMD treated part of periodontium [13], details of osteoinductive properties of this material can be very useful for practical usage. Also regeneration of bone and its enhancement are important for periodontal regeneration, especially the knowledge about human bone marrow cells behavior under application of EMP can be important for practical day by day usage in different clinical cases.
Aim
To investigate presence of osteoinductive properties of “Emdogain” (Straumann) and evaluate the influence on osteogenous progenitor cells-colony-forming fibroblast units (CFFU) of human bone-marrow ex vivo.
Materials and methods
Cloning of CFFU of human bone marrow was provided according to methodic of Fridenshtein and Lalikina [53] in modification of Astachova [54]. For investigation the cancellous iliac bone was taken from healthy patients out of inflammatory and degenerative-dystrophic lesions during orthopedic surgical operation.
The cancellous bone for investigation was take in the conditions of operating room, put into container with feeding solution “199”. Further processing of the collected material was provided in laboratory box under sterile conditions. The cell cloning procedure was taken under standard conditions during 14 days without changing of feeding solution in Ru-containers (culture containers) under temperature of 37°C in gaseous blend containing 5% of CO2 in atmosphere oxygen with usage of lethally irradiated rabbit bone marrow cells in role of feeder.
For investigation of direct action of “Emdogain” (Straumann), “Pre-Gel” (Straumann) ex vivo 4 series experiments of cloning CFFU of human bone marrow were provided. In 1st experimental group into culture container during explantation of bone marrow cells “Pref-Gel” (Straumann) was added. In 2nd experimental group “Pref-Gel” (Straumann) and “Emdogain” (Straumann) were added into culture container during explantation of bone marrow cells. In 3rd experimental group only “Emdogain” (Straumann) was added into culture container. In 4th experimental group, as a control group, the cloning of CFFU was provided without adding of any additional material.
The regeneration potential of bone tissue was assessed according to value of cloning effectiveness of CFFU of human bone marrow among 105 nuclear containing cells. The cloning effectiveness was assessed according formula:
Where K - means amount of colonies that grew up in cultural container x 105; N - means amount of cells that were explanted into cultural container.
The statistics was provided according computer program analysis “Statistica”. The middle values presented as M±m, where M - the middle meaning of value, m - standard deviation of middle value meaning.
The 4 experimental series of CFFU cloning of human bone-marrow were provided: 1 group - with adding of etching gel “Pref-Gel”; 2 group - with adding of “Pref-Gel” with “Emdogain”; 3 - with adding of “Emdogain” only and 4 - without adding of any preparations (control). The action was evaluated according to cloning effectiveness of CFFU of human bone-marrow among 105 nucleus containing cells. 9 experimental and 6 control colonies were cultivated.
Results and Discussion
In 1st and 2nd experimental groups the growth of stromal fibroblasts wasn’t detected. The effectiveness of CFFU cloning = 0. In 3rd group in the mean 145 colonies of CFFU grew up with cloning effectiveness of 15,10 ± 0,95 among 105 nucleus-containing cells. In control group in the mean 120 colonies of CFFU grew up. The cloning effectiveness was 12,48 ± 1,24 among 105 nucleus-containing cells. Ad oculus the colonies from control and investigated groups weren’t differ from each other. “Emdogain” (Straumann) on 20,8% in comparison with control group, enhanced amount of CFFU colonies in bone-marrow, increasing specific gravity of multilayer colonies, giving evidence about its osteoinductive properties.
Conclusion
The presented investigation showed the direct action of “Emdogain” (Straumann) with additional components on osteogenous progenitor CFFU of human bone marrow of iliac bone ex vivo, the certain details of action were determined. The obtained data can be useful for regenerative periodontal surgical procedures. Adding of “Pref-gel” (Straumann) for etching procedure into culture of stromal fibroblasts of human bone marrow completely depress the colonies growth. “Pref-Gel” and combination of “Pref-gel” with “Emdogain” completely depresses proliferation and differentiation of CFFU in human bone marrow ex vivo. The cloning activity was absent. This means that EDTA for etching stage application, which is chemically active component of “Pref-Gel” (Straumann) influences negatively on vital cells of bone tissue that must be taken into consideration during practical usage. During application providing periodontal surgical operations having exposed bone in area of bone pockets, it may influence on vital cells leading to their death in area of operating periodontal wound.
Knowing the fact that according proper instruction guidelines it is recommended to etch the root surface previously to “Emdogain” application, it is need able to notice the importance, which can be questionable, of combined usage of etching procedure with EDTA and afterwards EMD application. This situation can be explained with difficulties to prevent entering of etching gel-like EDTA into deep bone pocket with exposed bone after preparation procedures including influence of exposition timing. Also this detail is important while the bone pockets are containing and deep where in most of cases its need able to fill all pocket in aim to prevent root surface against blood coverage. From the other hand the obligatory usage of etching of root surface due to EDTA application is under contradiction and it is important to notice that additional protective measures for bone preservation against EDTA penetration should be provided otherwise.
In contrast “Emdogain” (Straumann) alone enhances growth of CFFU colonies in bone marrow due to increased specific gravity of multilayer colonies, giving evidence about existence of osteoinductive properties. Even if the grew colonies from CFFU were not differ from each other in different investigated groups, “Emdogain” (Straumann) strongly enhanced their formation in 20,8%, that can be assessed as strongly enhanced.
Profound knowledge about details of influence of EMD on bone tissue may provide developing of more precise and accurate periodontal surgical procedures with less invasion and tissue preservation. This may provide additional EMD usage in dental practice dealing with bone regenerative procedures, periodontal wound healing enhancement.
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Copyright: © 2014 Biloklytska G F., et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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PMID: 28936490 [PubMed]

PMCID: PMC5604476


EC Neurology
Examining those Meeting IOM Criteria Versus IOM Plus Fibromyalgia

PMID: 28713879 [PubMed]

PMCID: PMC5510658


EC Neurology
Unilateral Frontosphenoid Craniosynostosis: Case Report and a Review of the Literature

PMID: 28133641 [PubMed]

PMCID: PMC5267489


EC Ophthalmology
OCT-Angiography for Non-Invasive Monitoring of Neuronal and Vascular Structure in Mouse Retina: Implication for Characterization of Retinal Neurovascular Coupling

PMID: 29333536 [PubMed]

PMCID: PMC5766278


EC Neurology
Longer Duration of Downslope Treadmill Walking Induces Depression of H-Reflexes Measured during Standing and Walking.

PMID: 31032493 [PubMed]

PMCID: PMC6483108


EC Microbiology
Onchocerciasis in Mozambique: An Unknown Condition for Health Professionals.

PMID: 30957099 [PubMed]

PMCID: PMC6448571


EC Nutrition
Food Insecurity among Households with and without Podoconiosis in East and West Gojjam, Ethiopia.

PMID: 30101228 [PubMed]

PMCID: PMC6086333


EC Ophthalmology
REVIEW. +2 to +3 D. Reading Glasses to Prevent Myopia.

PMID: 31080964 [PubMed]

PMCID: PMC6508883


EC Gynaecology
Biomechanical Mapping of the Female Pelvic Floor: Uterine Prolapse Versus Normal Conditions.

PMID: 31093608 [PubMed]

PMCID: PMC6513001


EC Dental Science
Fiber-Reinforced Composites: A Breakthrough in Practical Clinical Applications with Advanced Wear Resistance for Dental Materials.

PMID: 31552397 [PubMed]

PMCID: PMC6758937


EC Microbiology
Neurocysticercosis in Child Bearing Women: An Overlooked Condition in Mozambique and a Potentially Missed Diagnosis in Women Presenting with Eclampsia.

PMID: 31681909 [PubMed]

PMCID: PMC6824723


EC Microbiology
Molecular Detection of Leptospira spp. in Rodents Trapped in the Mozambique Island City, Nampula Province, Mozambique.

PMID: 31681910 [PubMed]

PMCID: PMC6824726


EC Neurology
Endoplasmic Reticulum-Mitochondrial Cross-Talk in Neurodegenerative and Eye Diseases.

PMID: 31528859 [PubMed]

PMCID: PMC6746603


EC Psychology and Psychiatry
Can Chronic Consumption of Caffeine by Increasing D2/D3 Receptors Offer Benefit to Carriers of the DRD2 A1 Allele in Cocaine Abuse?

PMID: 31276119 [PubMed]

PMCID: PMC6604646


EC Anaesthesia
Real Time Locating Systems and sustainability of Perioperative Efficiency of Anesthesiologists.

PMID: 31406965 [PubMed]

PMCID: PMC6690616


EC Pharmacology and Toxicology
A Pilot STEM Curriculum Designed to Teach High School Students Concepts in Biochemical Engineering and Pharmacology.

PMID: 31517314 [PubMed]

PMCID: PMC6741290


EC Pharmacology and Toxicology
Toxic Mechanisms Underlying Motor Activity Changes Induced by a Mixture of Lead, Arsenic and Manganese.

PMID: 31633124 [PubMed]

PMCID: PMC6800226


EC Neurology
Research Volunteers' Attitudes Toward Chronic Fatigue Syndrome and Myalgic Encephalomyelitis.

PMID: 29662969 [PubMed]

PMCID: PMC5898812


EC Pharmacology and Toxicology
Hyperbaric Oxygen Therapy for Alzheimer's Disease.

PMID: 30215058 [PubMed]

PMCID: PMC6133268


News and Events


August Issue Release

We always feel pleasure to share our updates with you all. Here, notifying you that we have successfully released the August issue of respective journals and can be viewed in the current issue pages.

Submission Deadline for September Issue

Ecronicon delightfully welcomes all the authors around the globe for effective collaboration with an article submission for the September issue of respective journals. Submissions are accepted on/before August 21, 2020.

Certificate of Publication

Ecronicon honors with a "Publication Certificate" to the corresponding author by including the names of co-authors as a token of appreciation for publishing the work with our respective journals.

Best Article of the Issue

Editors of respective journals will always be very much interested in electing one Best Article after each issue release. The authors of the selected article will be honored with a "Best Article of the Issue" certificate.

Certifying for Review

Ecronicon certifies the Editors for their first review done towards the assigned article of the respective journals.

Latest Articles

The latest articles will be updated immediately on the articles in press page of the respective journals.

Immediate Assistance

The prime motto of this team is to clarify all the queries without any delay or hesitation to avoid the inconvenience. For immediate assistance on your queries please don't hesitate to drop an email to editor@ecronicon.uk