|Year : 2020 | Volume
| Issue : 4 | Page : 301-310
Biomimetic properties of engineered periodontal ligament/cementum in dental implants
Anil Mathew1, Anna Serene Babu1, Arun Keepanasseril2
1 Department of Prosthodontics, Amrita School of Dentistry, Amrita Vishwa Vidyapeetham, Kochi, Kerala, India
2 Amrita Institute of Medical Sciences, Amrita Vishwa Vidyapeetham, Kochi, Kerala, India
|Date of Submission||14-Mar-2020|
|Date of Decision||13-Jul-2020|
|Date of Acceptance||19-Jul-2020|
|Date of Web Publication||20-Dec-2020|
Dr. Anil Mathew
Department of Prosthodontics, Amrita School of Dentistry, Amrita Institute of Medical Sciences, Amrita Vishwa Vidyapeetham, Ponekkara P.O, Kochi, Kerala
Source of Support: None, Conflict of Interest: None
| Abstract|| |
The conventional concept of osseointegrated dental implants based on direct connection to alveolar bone lacks a structured periodontal ligament (PDL) as in natural tooth. This limits the physiologic and functional efficiency of the implant in cushioning occlusal overload, orthodontic tooth movement, and proprioception. Development of bio-mimetic implants that can satisfy the bio-functional requirements of the natural tooth will be an innovative approach and preliminary researches in this area has been reported. This review includes in vivo studies which reported structural features and functional efficiency of an artificial PDL or cementum developed around dental implants. The electronic search identified 12 animal studies and one human trial which utilized retained or adjacent natural tooth roots, exogenous scaffold materials, dental progenitor cells derived from PDL of extracted tooth root as PDL substitutes. The result of the review is dominated by bio-hybrid implants that used dental follicles separated on the particular embryonic day and cell sheets from immortalized human cells. A summary of the currently available research on artificial PDL/cementum around dental implants highlights the potential need of autologous cell-derived tissues to bioengineer a fully functional implant design
Keywords: Artificial cementum, artificial periodontal ligament, biohybrid implants, bioengineered periodontal ligament, bio-implant, dental implants
|How to cite this article:|
Mathew A, Babu AS, Keepanasseril A. Biomimetic properties of engineered periodontal ligament/cementum in dental implants. Contemp Clin Dent 2020;11:301-10
| Introduction|| |
Osseointegrated dental implants are the treatment of choice for rehabilitation of missing teeth availing a paradigm shift from conventional removable or fixed prosthetic options to tooth-root like fixture secured in the jaw bone. However, the absence of periodontal ligament (PDL) attachment around the implant hinders its biological function efficiency. PDL in natural tooth forms a fibrous connective tissue between the natural tooth root and surrounding bone. The periodontal tissue, which includes PDL, cementum, and alveolar bone, has an essential physiological role in cushioning high occlusal loads, orthodontic tooth movement in conjunction with bone remodeling and ability to perceive noxious stimuli., The ligament acts as a viscoelastic ” shock absorber” by diminishing the magnitude of the occlusal force and dissipating stress applied to the surrounding bone. The excessive occlusal load applied to a rigidly fixed dental implant, which lacks PDL, thus directly imposes stress on the bone interface presenting implant mobility, a clinical sign of failure. Dental implants are also known to be susceptible to infection owing to the absence of potential cellular defense mechanism of PDL. To compensate for the negative effects of occlusal overload, concept of implant protected occlusion was introduced, which incorporates certain clinical guidelines to establish an occlusal scheme and thus improve implant longevity. But this type of individualized occlusion needs to be re-evaluated and adjusted frequently. A controversy also exists whether the ” nonmobile” implant has the potential to bear the load of the prosthesis when splinted to a ” mobile” natural teeth. The osseointegrated dental implant limits its application in the growing jaw bone and in patients with severe bone defects. All these reported drawbacks necessitate the need for advances in dental implant structure that can simultaneously satisfy physiological and functional requisites. As a critical solution, apposite cell sources in adult tissue and pertinent bioengineering advancements need to be pooled to regenerate an artificial periodontium around the implant. The holy grail is to develop a biomimetic implant that can satisfy the bio-functional requirements of a natural tooth.
The emerging field of regenerative dentistry aims to develop novel fibrous tooth implant connections using dental follicle stem cells, bioengineered PDL, transplanted PDL cell sheets derived from PDL-derived stem cells (PDLSCs) and biodegradable scaffolds.,,
This scoping review provides an insight into available techniques and biomaterials used in potential periodontal tissue regeneration with emphasis on the most recent outcomes and future avenues.
| Methodology|| |
Information sources and search criteria
The initial search performed could not find any Mesh terms or standardized vocabularies catering to our stated aims. Therefore, a group of keywords were manually generated from the previous relevant articles and their citations. These were submitted to an expert panel, which conducted a focus group and selected the terms used in the literature search for this review. Based on this, an electronic search of the literature in English-language published from 1989 to 2018 was undertaken on January 2, 2019, in MEDLINE via Ovid interface. The search strategy used is mentioned in [Table 1]. A manual unstructured search was also conducted to identify additional citations.
Study eligibility criteria
Studies for this were included if they were published in English and satisfied all of the following criteria:
- Reported development of either artificial PDL or artificial cementum
- In vivo studies dealing with transplantation of the artificial PDL into the bone
- Used titanium dental implants or implant surfaces
- Described the microscopic structure of newly formed PDL or functional evaluation or response to noxious stimuli.
We excluded in vitro studies, hypothetical studies, commentaries, and systematic reviews after including relevant primary articles from the reviews. Studies which discussed periodontal defect regeneration and bone regeneration were excluded. Studies using extra oral craniofacial implants and bone implants were also excluded.
Citations retrieved in the database searches were screened in duplicate by two blinded reviewers. In the initial stage, title and abstract screening were performed, followed by full-text screening in the next phase. Disagreements and conflicts during all the screening stages were resolved by a third adjudicator. Screening of eligible studies, verification of their potential relevance, and full-text reading were conducted independently by AM and ASB.
Data extraction was performed in duplicate by the two reviewers, and the third reviewer resolved the conflict. Extracted data included author, country, institution, study type, number, and type of implant used, intervention, i.e., artificial PDL or cementum incorporated, site and method of transplantation, study design, the animal used, duration, test or analysis, features of newly formed PDL, microscopic structure, functional evaluation, and response to noxious stimuli.
| Results|| |
The electronic and manual search performed in the initial stage yielded 1395 records, of which 46 were identified as duplicates and removed. After screening of titles and abstracts, 1305 articles were found irrelevant and rejected as the majority of them discussed periodontal tissue regeneration, including alveolar bone, and studies restricted to bone defect management. The remaining 44 articles were considered eligible for full-text reading out of which 12 articles satisfied the defined inclusion criteria, Details on excluded studies and reasons for exclusion are depicted in the PRISMA flowchart [Figure 1].
Description of included studies
A detailed description of the included studies is summarized in [Table 2]. All the 12 included studies were animal studies, among which one study followed a human trial. Three studies used canine models,,, two studies primates,, one swine model, and one study utilized the goat model. Five were rodent studies,,,, and one was a rabbit study. Commercially pure titanium implants were considered as the study population in all the included studies. Five studies used HA (hydroxyapatite) coated implants,,,,, with an additional platinum coating applied in one study. Sandblasted and acid-etched (SLA) implants were placed in one study. Plasma sprayed Ti implants were selected in two studies,, one of which reported a hollow cylindrical design.
Structural features of the periodontal tissue
The preliminary studies utilized PDL derived from the retained root and adjacent tooth.,, Buser et al. analyzed cementum formation on plasma-sprayed dental implants transplanted in regions of the alveolar process with retained apical root portions for 12 months. The results revealed the formation of a hard tissue layer on implant surface, which depicts direct continuity with newly formed ” root” cementum taking its source from PDL cells of adjacent roots. A soft-tissue space with dimension and structure similar to PDL space with abundant connective tissue fibers perpendicular to implant surface confirmed the formation of a true PDL around titanium implants. A similar study by Urabe et al. with his co-workers compared the morphogenetic behavior of periodontium on titanium alloy (TA), and hydroxyapetite (HA) coated implants placed in the inter premolar spaces on mandible in contact with the roots of adjacent premolars and canines. An obvious fibrous connective tissue and mature calcified cementum-like tissue in continuity with the PDL of adjacent teeth were noted. The histomorphometric analysis highlighted uniformly aligned fibrous connective tissue on HA-coated implants; while they were nonuniform with no collagen fibers inserted on TA implants. Warrer et al. established that the progenitor cells for cementum formation derived from the retained root PDL initiated the formation of new cementum layer along the implant surface with Sharpey's fibers oriented perpendicular to the surface.
Among the early studies, implantation of bone wedges sheathed by PMMA and [email protected] filamentous tissue presented surrounding granulation tissue and few giant cells in varying amounts of dense collagen bundles. Neo formed vessels were also identified owing to migration and proliferation of endothelial cells. Piattelli et al. proposed the placement of expanded poly tetrafluoroethylene (ePTFE) membranes on the crestal bone around the implant with an artificial bone defect, which resulted in proper healing with initial direct bone contact and good primary stability without any inflammation. Histological features revealed formation of root cementum described as ” osteocementum” or ” dystrophic cementum,” similar to the tissue found in the cementum-producing lesions of the jawbones. In the early 20s, investigations using cultured PDL cells retrieved from the mid-root surface of extracted natural tooth showed a thin layer of cementum-like tissue on 37% of implant surfaces with collagen fibers in the PDL–like tissue oriented perpendicular to the implant surface. Further, 50:50 poly DL-lactideco-glycolide (PLG) scaffolds as intervention demonstrated highly cellular and vascular newly formed collagen fibers with deposition of cementum like layer tightly adhered to implant serration surface by means of a connective tissue fiber bundle resembling Sharpey's fibers. Gault et al. and co workers in 2010 described the development and clinical application of a tissue-engineered ligament ” ligaplants,” a combination of PDL cells with implant biomaterial. Here the human PDL cells isolated from an extracted tooth were cultured in a bioreactor to get cell sheets that were rolled onto porous hydroxyapatite (HAP) implant discs and transplanted to animal model as host for cell culture. The ligaplant combined with canine-derived PDL cells with additional fibronectin coating, was then implanted into the jaw of human cell donors. Even though the primary fitting was loose to spare the PDL cell cushion, the bio-engineered ligaplants assured firm integration with the induction of new bone and favorable micro-movements within the bone. Palisades of elongated cells equivalent to normal PDL and newly formed layers resembling cementum were identified. Collagen fiber bundles inserted of the human source as detected by anti-human collagen serum. A report by Lin et al. in 2011 using rat PDL derived autologous dental progenitor cells (DPCs) with Matrigel, a three-dimensional biomatrix scaffold rich in necessary extracellular matrix components which presented granulation tissue at the bone-implant interface at 8 weeks and osseointegration achieved at 18 weeks with poor vascularization and organized PDL like tissue formation. In the recent literature, a novel fibrous connected implant that used embryonic dental follicle stem cells was established as a proof-of-concept for a next-generation bio-hybrid dental implant by Oshima et al. [Figure 2]. Here the implant was enveloped with embryonic day (ED) 18.5 tooth germ-derived dental follicle tissue (ED18.5-DF) and transplanted into an adult mouse. Lack of proper cementum formation on the implant surface reflected as a critical issue of biohybrid implants to construct a functional tissue similar to the natural periodontal tissue. This was approached by incorporating bone tissue with an osseointegrated dental implant as a substitute for cementum. HA-coated implants were transplanted and allowed to osseointegrate. The bone attached implants were then retrieved and wrapped in cultured rat PDL tissue and re-transplanted. These studies recognized PDL fiber structure comprised of transverse collagen fibers and longitudinal elastin fibers along with accurate cementum formation on the implant surface. Electron probe microanalysis revealed high concentrations of specific hard tissue elements such as calcium and phosphorus in cementum and alveolar bone, but not in the PDL region, as in natural tooth., The most recent literature in this review reported the use of immortalized human cells for the regeneration of live periodontium. The four cell types used as single or multi-layered cell sheet technique were immortalized human PDL (ihPDLs) cells, immortalized human cementoblasts (ihCEMs), human umbilical vein endothelial cells (HUVECs) and porcine-derived epithelial cell rests of Malassez (ERM). The transplanted cell sheets formed PDL-like connective tissue aligned vertical and parallel to the implant surface. The presence of blood vessels and the existence of oxytalan fibers were confirmed. The PDL markers, periostin, and fibrillin 1 staining were positive around the PDL-like tissue with signs of alveolar bone remodeling.
|Figure 2: Schematic representation of bio-hybrid dental implant model|
Click here to view
Functional efficiency of periodontal ligament
Functional analysis of the PDL was validated in five studies.,,,, Two studies which demonstrated the development of bio-hybrid implant analyzed whether the application of mechanical force could bring about orthodontic movement in the experimental model. Colony-stimulating factor-1 (Csf-1) used as a marker of osteoclastogenesis and osteocalcin (Ocn) were observed on the compression and tension sides, respectively. The results displayed movement of bio-hybrid implant similar to natural teeth with successful bone remodeling through the appropriate orientation of osteoclasts and osteoblasts when exposed to mechanical stress., The functional quality analysis of ihCEMs presented expression of mineralization-related genes, whereas the ihPDLs expressed collagen type I and PDL specific marker. The results of the preliminary study, which utilized PMMA Dacron material, reported unaffected elastic properties when subjected to shear stress. Mechanical resistance around ligaplant inserted in human jaw evaluated through mobility tests presented scores similar to the natural tooth when compared to osseointegrated implants, which was zero.
Ability to perceive noxious stimulation
The response of the novel fibrous periodontal connection to noxious stimuli was evaluated in two studies included in the review with the engrafted bio-hybrid implant as the intervention. The detection of Antineurofilament (NF)-immunoreactive nerve fibers in the newly formed PDL of bio-hybrid implant was one of the proof of evidence. There was an increase in C-Fosimmunoreactive neurons at 2 h after orthodontic treatment, which are usually noticed in the noxious mechanical and chemical trigger of the intraoral receptive fields, and also in the natural tooth.,
| Discussion|| |
The conventional concept of implant osseointegration lacks the support of PDL, which provides cushioning effect to heavy occlusal load. Since PDL tissue engineering is budding as a solution for periodontal treatment, its application in conjunction with a dental implant can be a promising approach to meet the existing shortcomings related to mechanical and sensory efficiency of osseointegrated implants. This review summarizes the current body of evidence in the development of a biomimetic implant by incorporating an artificial PDL.
Our review identified a paradigm shift from the utilization of retained or adjacent natural tooth roots as PDL,, to exogenous scaffold materials like PLG expanded ePTFE membranes and Dacron tissue in the preliminary works.,, Then DPCs derived from PDL of the extracted tooth root surface of human or animal models were cultured and implanted in the next decade studies.,, Current researches show a striking trend towards bio-hybrid implants which used dental follicles separated on the particular embryonic day and the latest study using cell sheets from immortalized human cells.,,
The structural analysis of the newly formed PDL derived from retained or adjacent roots, and rat/human extracted PDL cells propose that the collagen fibers aligned perpendicular to the implant might enhance cementum formation on the surface. Formation of connective tissue fiber bundle along with the presence of neo-formed blood vessels was confirmed in studies using bioengineered scaffolds and immortalized human cells. Few interventions proved to compensate for both mechanical stress and sensory efficiency of natural PDL along with structural similarities exhibiting stable elastic properties to stress and response to mobility tests as that of the natural tooth.,,,, But an absolute functional recovery and stimuli perception is deficient in the simulated PDL formed around titanium implants, which exhibits the need for an apposite adult cell source to regenerate a more favorable autologous PDL.
The limitation of the current review can be stated as the possibility of missing relevant articles during the search due to a lack of standardized search terms. Another limitation is in vitro studies excluded during the initial search, as some of them might have reported detailed information regarding the cellular morphology of newly formed PDL. These deficiencies might prevent the readers from absorbing an apparent depiction of the focused topic.
The current researches still focus on laboratory experiments with animal models except for one human study with ligaplants, which has surpassed the basic mobility test. Considering the anatomic and morphological limitation of animals when compared to humans, specific long term clinical trials are critical in this field. Assessment of mechanical and sensory response of dental implants with artificial PDL implanted in human jaws to simulate the physiologic functions of natural tooth still remains the need of the hour. The ongoing and next-generation regenerative therapies for dental diseases, tooth regeneration, and periodontal regeneration have used stem cells derived from various human tissues.,,, the identification and application of the same in PDL regeneration around dental implants with biofunctional characteristics will facilitate the development of clinically successful osseointegrated implants. Even though the decisive advantages of ligaplants were hopefully reviewed as a new dimension, factors like complex preparation process, caution about temperature, and duration of culturing, nonperiodontal cells obtained due to culturing failure and unpredictable host acceptance questions the feasibility of ligaplants. Some of the recent reviews updated the application of cell sheet engineering to form new cementum around dental implants by transplantation of PDL stem cells (PDLSCs) as an effective and safe approach., Thus, the schema for future research demands a combination of bioengineering techniques optimized with suitable adult stem cells as a potential candidate for the regenerative therapies allied with current dental implant design.
The future clinical utility of artificial PDL implies periodontal tissue regeneration, thus avoiding bone grafting in periodontal bone defects and successful restoration of perception through proper nerve innervations. Therefore these hybrid live dental implants due to its rich vascularity and cellular defense mechanism can be utilized in clinical conditions demanding repair of periodontium and areas susceptible to infections where osseointegrated implants are not indicated owing to its different wound healing mechanism. Another potential clinical application of implants with PDL pertains to orthodontic tooth movement to optimal positions in growing patients as the bone turnover in conventional implants with direct bone contact is not an adaptive process.
A tissue engineering approach is the foundation to this regenerative field that utilize pluripotent progenitor cells like PDL derived cells, mesenchymal stem cell isolation, biodegradable polymer structures, and recent advances being gene therapy using vectors or direct delivery technique.
To summarize, there is certainly an emerging body of evidence authenticating the possibility of ligamentous attachment around dental implants satisfying the concept of functional and biological tooth replacement in infection susceptible clinical scenario and orthodontic movement perspective. A key concern has been the practical application concerning the feasibility and time consumption in the clinical setting.
| Conclusion|| |
Precise work to refine techniques using DPCs and human-derived cells integrated with bioengineered scaffolds need to be developed and experimented as clinical trials. The concept of artificial PDL has the potential to restore missing natural teeth using oral implants augmented with autologous cell-derived bioengineered tissues. This review proposes the future scope for an absolute prosthetic rehabilitation using functional osseointegrated implants with bio physiologic performance simulating the missing teeth.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Alissa R, Oliver RJ. Influence of prognostic risk indicators on osseointegrated dental implant failure: A matched case-control analysis. J Oral Implantol 2012;38:51-61.
Willis RD, DiCosimo CJ. The absence of proprioceptive nerve endings in the human periodontal ligament: The role of periodontal mechanoreceptors in the reflex control of mastication. Oral Surg Oral Med Oral Pathol 1979;48:108-15.
Avery JK. Oral development and histology. In: Steele PF, editor. Oral development and histology. New York: Thieme Press; 2002. p. 225-42.
Proffit WR, Fields HW, Sarver DM. Contemporary Orthodontics. 4th
ed. St. Louis: Mosby Elsevier; 2006. p. 77-83.
Shimono M, Ishikawa T, Ishikawa H, Matsuzaki H, Hashimoto S, Muramatsu T, et al
. Regulatory mechanisms of periodontal regeneration. Microsc Res Tech 2003;60:491-502.
Esposito M, Thomsen P, Ericson LE, Sennerby L, Lekholm U. Histopathologic observations on late oral implant failures. Clin Implant Dent Relat Res 2000;2:18-32.
Schou S, Holmstrup P, Stoltze K, Hjørting-Hansen E, Kornman KS. Ligature-induced marginal inflammation around osseointegrated implants and ankylosed teeth. Clin Oral Implants Res 1993;4:12-22.
Misch CE, Bidez MW. Occlusal considerations for implant-supported prosthesis: Implant protective occlusion. In: Dental implant prosthetics. St. Louis: Elsevier/Mosby; 2005. p. 472-507.
Burns DR, Beck DA, Nelson SK. A review of selected dental literature on contemporary provisional fixed prosthodontic treatment: Report of the committee on research in fixed prosthodontics of the academy of fixed prosthodontics. J Prosthet Dent 2003;90:474-97.
Ikeda E, Tsuji T. Growing bioengineered teeth from single cells: potential for dental regenerative medicine. Expert Opin Biol Ther 2008;8:735-44.
Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S, Brahim J, et al
. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 2004;364:149-55.
Sasai, Y. Next-generation regenerative medicine: Organogenesis from stem cells in 3D culture. Cell Stem Cell 2013;12:520-30.
Gault P, Black A, Romette JL, Fuente F, Schroeder K, Thillou F, et al
. Tissue-engineered ligament: Implant constructs for tooth replacement. J Clin Periodontol 2010;37:750-8.
Choi BH. Periodontal ligament formation around titanium implants using cultured periodontal ligament cells: A pilot study. Int J Oral Maxillofac Implants 2000;15:193-6.
Urabe M, Hosokawa R, Chiba D, Sato Y, Akagawa Y. Morphogenetic behaviour of periodontium on inorganic implant materials: An experimental study of canines. J Biomed Mater Res 2000;49:17-24.
Buser D, Warrer K, Karring T, Stich H. Titanium implants with a true periodontal ligament: An alternative to osseointegrated implants? Int J Oral Maxillofac Implants 1990;5:113-6.
Warrer K, Karring T, Gotfredsen K. Periodontal ligament formation around different types of dental titanium implants. I. The self-tapping screw type implant system. J Periodontol 1993;64:29-34.
Piattelli A, Cordioli GP, Passi P, Trisi P. Formation of dental hard tissues and periodontal ligament around titanium implants after tooth-bud injury: A pilot study. Int J Oral Maxillofacial Implants 1994;9:417-21.
Marei MK, Saad MM, El-Ashwah AM, El-Backly RM, Al-Khodary MA. Experimental formation of periodontal structure around titanium implants utilizing bone marrow mesenchymal stem cells: A pilot study. J Oral Implantol 2009;35:106-29.
Lin Y, Gallucci GO, Buser D, Bosshardt D, Belser UC, Yelick PC. Bioengineered periodontal tissue formed on titanium dental implants. J Dent Res 2011;90:251-6.
Oshima M, Inoue K, Nakajima K, Tachikawa T, Yamazaki H, Isobe T, et al
. Functional tooth restoration by next-generation bio-hybrid implant as a bio-hybrid artificial organ replacement therapy. Sci Rep 2014;4:6044.
Nakajima K, Oshima M, Yamamoto N, Tanaka C, Koitabashi R, Inoue T, et al
. Development of a functional bio hybrid implant formed from periodontal tissue utilizing bioengineering technology. Tissue Eng Part A 2016;22:1108-15.
Lee DJ, Lee JM, Kim EJ, Takata T, Abiko Y, Okano T, et al
. Bio-implant as a novel restoration for tooth loss. Sci Rep 2017;7:7414.
Caiazza S, Taruscio D, Ciaralli F, Crateri P, Chistolini P, Bedini R, et al
. Evaluation of an experimental periodontal ligament for dental implants. Biomaterials 1991;12:474-8.
Volponi AA, Pang Y, Sharpe PT. Stem cell-based biological tooth repair and regeneration. Trends Cell Biol 2010;20:715-22.
Yang B, Qiu Y, Zhou N, Ouyang H, Ding J, Cheng B, et al
. Application of stem cells in oral disease therapy: Progresses and perspectives. Front Physiol 2017;8:197.
Bassir SH, Wisitrasameewong W, Raanan J, Ghaffarigarakani S, Chung J, Freire M, et al
. Potential for stem cell-based periodontal therapy. J Cell Physiol 2016;231:50-61.
Xu XY, Li X, Wang J, He XT, Sun HH, Chen FM. Concise review: Periodontal tissue regeneration using stem cells: Strategies and translational considerations. Stem Cells Transl Med 2019;8:392-403.
Garg H, Deepa D. Bioengineered periodontal ligament: Ligaplants, a new dimension in the field of implant dentistry Mini review. J Oral Res Rev 2018;10:92. [Full text]
Iwasaki K, Washio K, Meinzer W, Tsumanuma Y, Yano K, Ishikawa I. Application of cell-sheet engineering for new formation of cementum around dental implants. Heliyon 2019;5:e01991.
Washio K, Ishikawa I, Yano K, Iwasaki K, Tumanuma Y. Innovative potential of periodontal ligament cell sheet engineering in functional implant therapy. Innovations Tissue Eng Regen Med 2018;1:000507.
[Figure 1], [Figure 2]
[Table 1], [Table 2]