Hengameh Bakhtiar1, Mohammad Pezeshki-Modaress2, Zahra Kiaipour3, Mahdieh Shafiee3, Mohammad Reza Ellini3, Amir Mazidi3, Sarah Rajabi4, Soheila Zamanlui Benisi3, Seyed Naser Ostad5, Kerstin Galler6, Pardis Pakshir7, Amir Azarpazhooh8, Anil Kishen9. 1. Department of Endodontics, Faculty of Dentistry, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran; Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada; Stem Cell Research Center, Tissue Engineering and Regenerative Medicine Institute, Tehran Central Branch, Islamic Azad University, Tehran, Iran. 2. Burn Research Center, Iran University of Medical Sciences, Tehran, Iran. 3. Stem Cell Research Center, Tissue Engineering and Regenerative Medicine Institute, Tehran Central Branch, Islamic Azad University, Tehran, Iran. 4. Department of Cell Engineering, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran. 5. Department of Toxicology-Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran. 6. Department of Conservative Dentistry and Periodontology, University Hospital Regensburg, Regensburg, Germany. 7. Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada. 8. Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada; Clinical Epidemiology and Health Care Research, Institute of Health Policy, Management and Evaluation, University of Toronto, Toronto, Ontario, Canada; Department of Dentistry, Mount Sinai Hospital, Toronto, Ontario, Canada. 9. Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada; Department of Dentistry, Mount Sinai Hospital, Toronto, Ontario, Canada. Electronic address: Anil.Kishen@dentistry.utoronto.ca.
Abstract
OBJECTIVE: Recent studies suggest xenogeneic extracellular matrices as potential regenerative tools in dental pulp regeneration. This study aimed to fabricate and characterize a novel three-dimensional macroporous pulp-derived scaffold that enables the attachment, penetration, proliferation and differentiation of mesenchymal stem cells. METHOD: Bovine pulp was decellularized and characterized with histological and DNA content methods. This scaffold was prepared using finely milled lyophilized decellularized pulp extracellular matrix (ECM) digested with pepsin. Three different concentrations of ECM (1.50, 2.25 and 3.00mg/ml) were freeze-dried and were tested with/without chemical crosslinking. The specimens were subjected to physicochemical characterization, cell viability and quantitative real time polymerase chain reaction assessments with human bone marrow mesenchymal stem cells (hBMMSCs). All scaffolds were subcutaneously implanted in rats for two weeks and histological and immunostaining analyses were performed. RESULTS: Histological and DNA analysis confirmed complete decellularization. All samples demonstrated more than 97% porosity and 1.50mg/ml scaffold demonstrated highest water absorption. The highest cell viability and proliferation of hBMMSCs was observed on the 3.00mg/ml crosslinked scaffolds. The gene expression analysis showed a significant increase of dmp-1 and collagen-I on 3.00mg/ml crosslinked scaffolds compared to the other scaffolds. Histological examination of subcutaneous implanted scaffolds revealed low immunological response, and enhanced angiogenesis in cross-linked samples compared to non-crosslinked samples. SIGNIFICANCE: The three-dimensional macroporous pulp-derived injectable scaffold developed and characterized in this study displayed potential for regenerative therapy. While the scaffold biodegradability was decreased by crosslinking, the biocompatibility of post-crosslinked scaffold was significantly improved.
OBJECTIVE: Recent studies suggest xenogeneic extracellular matrices as potential regenerative tools in dental pulp regeneration. This study aimed to fabricate and characterize a novel three-dimensional macroporous pulp-derived scaffold that enables the attachment, penetration, proliferation and differentiation of mesenchymal stem cells. METHOD:Bovine pulp was decellularized and characterized with histological and DNA content methods. This scaffold was prepared using finely milled lyophilized decellularized pulp extracellular matrix (ECM) digested with pepsin. Three different concentrations of ECM (1.50, 2.25 and 3.00mg/ml) were freeze-dried and were tested with/without chemical crosslinking. The specimens were subjected to physicochemical characterization, cell viability and quantitative real time polymerase chain reaction assessments with human bone marrow mesenchymal stem cells (hBMMSCs). All scaffolds were subcutaneously implanted in rats for two weeks and histological and immunostaining analyses were performed. RESULTS: Histological and DNA analysis confirmed complete decellularization. All samples demonstrated more than 97% porosity and 1.50mg/ml scaffold demonstrated highest water absorption. The highest cell viability and proliferation of hBMMSCs was observed on the 3.00mg/ml crosslinked scaffolds. The gene expression analysis showed a significant increase of dmp-1 and collagen-I on 3.00mg/ml crosslinked scaffolds compared to the other scaffolds. Histological examination of subcutaneous implanted scaffolds revealed low immunological response, and enhanced angiogenesis in cross-linked samples compared to non-crosslinked samples. SIGNIFICANCE: The three-dimensional macroporous pulp-derived injectable scaffold developed and characterized in this study displayed potential for regenerative therapy. While the scaffold biodegradability was decreased by crosslinking, the biocompatibility of post-crosslinked scaffold was significantly improved.