Synthetic Bone Substitute Engineered with Amniotic Epithelial Cells Enhances Bone Regeneration after Maxillary Sinus Augmentation

et al. (2013) Synthetic Bone Substitute Engineered with Amniotic Epithelial Cells Enhances Bone Regeneration after Maxillary Sinus Augmentation. PLoS ONE 8(5): e63256. doi:10.1371/journal.pone.0063256 Synthetic Bone Substitute Engineered with Amniotic Epithelial Cells Enhances Bone Regeneration after Maxillary Sinus Augmentation Barbara Barboni 0 Carlo Mangano 0 Luca Valbonetti 0 Giuseppe Marruchella 0 Paolo Berardinelli 0 Alessandra Martelli 0 Aurelio Muttini 0 Annunziata Mauro 0 Rossella Bedini 0 Maura Turriani 0 Raffaella Pecci 0 Delia Nardinocchi 0 Vincenzo Luca Zizzari 0 Stefano Tete` 0 Adriano Piattelli 0 Mauro Mattioli 0 Xiaoming He, The Ohio State University, United States of America 0 1 Department of Comparative Biomedical Science, University of Teramo , Teramo , Italy , 2 Department of Technologies and Health, Istituto Superiore di Sanita` , Rome , Italy , 3 Department of Surgical and Morphological Science, University of Insubria , Varese , Italy , 4 Department of Medical, Oral and Biotechnological Science, University ''G. d'Annunzio'' , Chieti , Italy , 5 Stem TeCh Group , Chieti , Italy Background: Evidence has been provided that a cell-based therapy combined with the use of bioactive materials may significantly improve bone regeneration prior to dental implant, although the identification of an ideal source of progenitor/stem cells remains to be determined. Aim: In the present research, the bone regenerative property of an emerging source of progenitor cells, the amniotic epithelial cells (AEC), loaded on a calcium-phosphate synthetic bone substitute, made by direct rapid prototyping (rPT) technique, was evaluated in an animal study. Material And Methods: Two blocks of synthetic bone substitute (,0.14 cm3), alone or engineered with 16106 ovine AEC (oAEC), were grafted bilaterally into maxillary sinuses of six adult sheep, an animal model chosen for its high translational value in dentistry. The sheep were then randomly divided into two groups and sacrificed at 45 and 90 days post implantation (p.i.). Tissue regeneration was evaluated in the sinus explants by micro-computer tomography (micro-CT), morphological, morphometric and biochemical analyses. Results And Conclusions: The obtained data suggest that scaffold integration and bone deposition are positively influenced by allotransplantated oAEC. Sinus explants derived from sheep grafted with oAEC engineered scaffolds displayed a reduced fibrotic reaction, a limited inflammatory response and an accelerated process of angiogenesis. In addition, the presence of oAEC significantly stimulated osteogenesis either by enhancing bone deposition or making more extent the foci of bone nucleation. Besides the modulatory role played by oAEC in the crucial events successfully guiding tissue regeneration (angiogenesis, vascular endothelial growth factor expression and inflammation), data provided herein show that oAEC were also able to directly participate in the process of bone deposition, as suggested by the presence of oAEC entrapped within the newly deposited osteoid matrix and by their ability to switch-on the expression of a specific bonerelated protein (osteocalcin, OCN) when transplanted into host tissues. - Funding: This work was supported by Tercas Foundation and by PRIN 20102011 (PRIN 20102ZLNJ5) financed by the Ministry of Education, University and Research (M.I.U.R.), Rome, Italy. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Bone regeneration in maxillary sinus is an essential condition for dental implants in atrophic posterior maxilla. Different strategies leading to the replacement of missing bone have been conventionally used for over 30 years [1,2]. Limited availability of autografts, and the risk of disease transmission by allo/xenografts, have increased the demand of synthetic bone substitutes, which have to reproduce the physical/chemical properties of native bone tissues in order to maximize osteointegration, osteoconduction and osteoinduction [2]. Calcium phosphate ceramics, such as hydroxyapatite (HA) and tricalcium-phosphate (TCP), are considered both suitable materials for bone reconstruction since they conjugate a high biocompatibility with an efficient osteoconductivity [3]. The porous architecture and the degree of interconnectivity are additional critical factors to determine the clinical success of biomaterials [4,5]. In fact, the chemical composition and architecture of biomaterials are both crucial to drive and stimulate bone healing and deposition. In order to mimic the structure of native bone and to ensure cell viability and function, the ideal scaffold should exhibit porosity at different length scales: nanoporosity, to allow molecule transport essential for any nutrition, waste removal and signaling; micro-porosity, to ensure cell migration and capillary formation; millimeter-wide porosity to incorporate nerves and blood vessels [6,7]. Scaffold porosity improves mechanical interlocking between the implanted biomaterial and the surrounding host bone [8,9], and positively influences the scaffold degradation rate. During the last few years, innovative technologies, such as three-dimensional (3D) printing and dispense-plotting, allowed to create scaffolds with a controlled 3D architecture [913], thus enhancing their biocompatibility [1417]. However, the latest generation of synthetic bone substitutes still requires a long time to regenerate a large amount of bone tissue thus limiting their surgical use in validated therapeutic protocols such as sinus augmentation [18,19]. Therefore, cell-based therapies are an emerging strategy to improve bone tissue healing and regeneration [2023]. In this context, increasing attention has been recently addressed to placental components and, in particular, to amnion as a possible reserve of stem/progenitor cells [2429]. Actually, the therapeutic use of amniotic membrane has been studied for decades. Davis first reported in 1910 the use of fetal membranes as surgical materials in skin transplantation performed on 550 patients [30]. Amniotic membranes showed anti-inflammatory [3133], antimicrobial [34], antifibroblastic [35] and low immunogenicity properties [36,37]. Several surgical applications for amniotic membranes have been reported, including their use as a biological dressing for the treatment of skin wounds, burn injuries and chronic leg ulcers, as well as in the treatment of tissue adhesion in surgical procedures and ocular burns [26]. More recently, amniotic membranes have been investigated as a possible source of stem/progenitor cells for therapeutic applications. Cells from mesenchymal and epithelial amniotic layers, amniotic mesenchymal stromal and amniotic epithelial cells (AEC), respectively, can be obtained without any ethical concerns, in large amounts and with validat (...truncated)