Combination of Bioactive Polymeric Membranes and Stem Cells for Periodontal Regeneration: In Vitro and In Vivo Analyses

RESEARCH ARTICLE Combination of Bioactive Polymeric Membranes and Stem Cells for Periodontal Regeneration: In Vitro and In Vivo Analyses Flávia Gonçalves1, Míriam Santos de Moraes2, Lorraine Braga Ferreira3, Ana Cláudia Oliveira Carreira4, Patrícia Mayumi Kossugue4, Letícia Cristina Cidreira Boaro3, Ricardo Bentini1, Célia Regina da Silva Garcia2, Mari Cleide Sogayar4,5, Victor Elias AranaChavez3, Luiz Henrique Catalani1* 1 Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brasil, 05508–000, 2 Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brasil, 05508–090, 3 Departamento de Biomateriais e Biologia Oral, Faculdade de Odontologia, Universidade de São Paulo, São Paulo, SP, Brasil, 05508–000, 4 NUCEL/NETCEM—Núcleo de Terapia Celular e Molecular, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brasil, 05360–130, 5 Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brasil, 05508–000 OPEN ACCESS Citation: Gonçalves F, de Moraes MS, Ferreira LB, Carreira ACO, Kossugue PM, Boaro LCC, et al. (2016) Combination of Bioactive Polymeric Membranes and Stem Cells for Periodontal Regeneration: In Vitro and In Vivo Analyses. PLoS ONE 11(3): e0152412. doi:10.1371/journal. pone.0152412 Editor: Salomon Amar, Boston University, UNITED STATES Received: August 5, 2015 Accepted: March 14, 2016 Published: March 31, 2016 Copyright: © 2016 Gonçalves 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. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The authors FG, RB and LHC would like to thank CNPq and FAPESP (2010/17698-2 and 2011/ 21442-6) for funding. Additionally, MM and CRSG acknowledge funding from CNPq and FAPESP (2011/51295-5), and ACOC, PMK and MCS acknowledge funding from BNDES, CNPq, FAPESP, FINEP, MCTI and MS-DECIT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. * Abstract Regeneration of periodontal tissues requires a concerted effort to obtain consistent and predictable results in vivo. The aim of the present study was to test a new family of bioactive polymeric membranes in combination with stem cell therapy for periodontal regeneration. In particular, the novel polyester poly(isosorbide succinate-co-L-lactide) (PisPLLA) was compared with poly(L-lactide) (PLLA). Both polymers were combined with collagen (COL), hydroxyapatite (HA) and the growth factor bone morphogenetic protein-7 (BMP7), and their osteoinductive capacity was evaluated via in vitro and in vivo experiments. Membranes composed of PLLA/COL/HA or PisPLLA/COL/HA were able to promote periodontal regeneration and new bone formation in fenestration defects in rat jaws. According to quantitative real-time polymerase chain reaction (qRT-PCR) and Alizarin Red assays, better osteoconductive capacity and increased extracellular mineralization were observed for PLLA/COL/ HA, whereas better osteoinductive properties were associated with PisPLLA/COL/HA. We concluded that membranes composed of either PisPLLA/COL/HA or PLLA/COL/HA present promising results in vitro as well as in vivo and that these materials could be potentially applied in periodontal regeneration. Introduction Periodontal disease is characterized by an inflammatory reaction and loss of dental support tissues [1]. Therefore, periodontal treatments primarily aim to eliminate the inflammatory process and to reestablish periodontal regeneration. In the early 1980s, the concept of guided tissue regeneration (GTR) was developed, which involves the use of an occlusive membrane to PLOS ONE | DOI:10.1371/journal.pone.0152412 March 31, 2016 1 / 21 Bioactive Polymeric Membranes for Periodontal Regeneration Competing Interests: The authors have declared that no competing interests exist. prevent the growth of epithelial tissue inside a periodontal defect, allowing the cells of the periodontal ligament to regenerate at the site [2]. The membrane was prepared from polytetrafluoroethylene, a biocompatible but non-degradable polymer. However, bacterial contamination and the need for a second surgery to remove the membrane were two of the primary concerns associated with this technique [3,4]. Currently, absorbable membranes made of collagen (COL) or biodegradable polyesters such as poly(L-lactide) (PLLA) and poly(lactic-coglycolic acid) (PLGA) are commercially available [2]. Although PLLA and PLGA have slower degradation [3] and better mechanical properties than COL membranes [5,6], these materials have low cell affinity [7]. COL membranes, which show higher cell affinity, are thus the most commonly used materials [7]. However, these membranes have inferior mechanical properties, collapse upon wetting, and have unpredictable degradation rates [8,9]. Despite the relatively satisfactory clinical performance of these materials [10,11], the results are not fully predictable, especially in cases with higher complexity, as in furcation lesions and one- or two-wall defects [12–14]. Therefore, new techniques, such as tissue engineering, have been proposed for periodontal regeneration. Tissue engineering uses scaffolds associated with biomolecules and differentiable stem cells to form appropriate dental supportive tissues [15]. Due to the structural intricacy of the periodontal ligament and the morpho-physiological diversity of its component tissues, the design of scaffolds for periodontal regeneration is highly complex. Efforts have been made to create multiphasic scaffolds in which a specific composition and/or structure are created for each of the tissues to be generated, namely, bone, periodontal ligament, and cement [9,16]. Several new materials have been evaluated in vitro [17–20], but few studies have evaluated the effectiveness of these materials in vivo with promising results [21–23]. In addition to its structure, the composition of the scaffold is extremely important in the engineering of periodontal tissues, as it determines the biocompatibility with host tissues and stimulates regeneration or inhibition of those tissues. The scaffold’s degradation rate should be proportional to the neoformation of regenerated tissue because rapid degradation can compromise neoformation [21], whereas slow degradation can promote encapsulation [24] or bone obstruction [25]. A combination of synthetic and natural polymers, such as COL [26], has been shown to be an interesting alternative for tissue engineering, as it combines the properties of both materials [6,27]. New materials, such as copolymers containing isosorbide succinate and L-lactide moieties, have shown promising surface properties, prom (...truncated)