Dental stem cell mediated bone tissue engineering using porcine model

Although bone has the potential to heal itself, genetic malformations, trauma and tumor surgery result in critical size defects in bone that need to be rehabilitated with clinical reconstruction procedures. The use of autogenous bone for the treatment of such defects is considered to be a golden standard (Von Wilmowsky et al., 2010). However, disadvantages, such as donor site morbidity, limited graft availability and morphological mismatch, make the reconstruction of these defects with autogenous bone challenging (Marolt, 2015). To eliminate the limitations of grafting and to establish a safer enhancement of bone formation, tissue-engineering approaches are employed. Tissue engineering is an interdisciplinary field that emerged as a result of the growing need for tissues and organs for transplantation and combines the knowledge of materials and cell biology for the development of tissue substitutes that act as "spare-parts" for the body. The primary goal of a bone tissue engineering scaffold is to provide an optimal environment for the natural healing mechanism of bone that is disrupted in critical-size defects (Schlegel et al., 2006). Tissue engineering includes 3 important parts: cells, scaffolds and biochemical cues. Bone scaffolds must be engineered to provide sufficient mechanical stability to the grafted area and support osteoconduction, osteoinduction and osteogenesis (Cypher & Grossman, 1996). Various biomaterials, such as natural or synthetic polymers, ceramics and composites, were used as tissue-engineering scaffolds to promote cell migration and differentiation, bone ingrowth and vascularization (Nair & Laurencin, 2007a). Also, bioactive molecules were added to enhance cell attachment, new bone formation and angiogenesis (Devescovi et al., 2008). In bone tissue engineering approach, cells with osteogenic potential are used to populate the scaffold for cell-mediated osteogenesis in combination with the osteoconductive effect of the scaffold (Kwan et al., 2007). For this purpose, mesenchymal stem cells (MSCs) derived from the bone marrow stroma have been used extensively in bone tissue engineering (Li et al., 2015; Liao et al., 2014). However, due to the complications related to bone marrow, such as surgical trauma caused by bone marrow harvesting procedures or bone marrow-related diseases, scientists focused on finding alternative resources of adult stem cells that require non-invasive or minimally invasive collection procedures. Recent studies have revealed the presence of adult stem cells in tissues of dental origin (Gronthos et al., 2000). In humans, third molars undergo organogenesis at around age 6 and do not completely develop until age 18. This means that undifferentiated cells remain in this tissue. Thus, tooth germ derived stem cells (TGSCs) are considered to be an ecto-mesenchymal source for isolating primitive stem cells that could differentiate into multiple lineages, including the osteogenic lineage (d'Aquino et al., 2008). Besides, the isolation of MSCs from dental tissue is easy, cost effective and does not raise additional safety and ethical concerns since they are obtained during regular orthodontic procedures. Stem cells of dental origin exhibit similar surface antigen profile with MSCs from other sources (Calikoglu Koyuncu et al., 2014). Stromal precursor antigen-1 (STRO-1), a surface antigen, is another surface antigen that is present only in a subpopulation of MSCs which are capable of differentiation into functional osteoblasts and include osteoprogenitors (Gronthos et al., 1994). Thus, selection of cells expressing STRO-1was thought to be an effective strategy to purify osteogenic cells from dental stem cells. Calcium phosphate based ceramics are frequently used as carrier materials in bone tissue engineering due to their osteoconductive properties and mechanical similarities with natural bone (Dubok et al., 2010; Samavedi et al., 2013). Various studies have utilized different ceramic materials either cultured with cells ex-vivo (Tsiridis et al., 2005) or combined with cells at the time of the surgery (Kokemueller et al., 2010). Although both approaches have different advantages in the clinical setting, the delivery of stem cells simultaneously with the alloplastic material during surgery seems to be an easier and feasible procedure to establish stem cell mediated bone healing. However, due to the poor volume stability and difficulty in handling the ceramic-based alloplasts (Spetzger et al., 2010), there is a need for additional materials that would facilitate the application of the graft while enhancing the survival and recruitment of stem cells. A biodegradable and bioactive polyethylene glycol (PEG) based hydrogel (MX10™, Straumann Holding AG, Switzerland) was introduced as novel carrier material that would have the ability to deliver proteins and cells at the defect site without damaging them. Thus, the delivery of stem cells using the PEG hydrogel and applying them in combination with a ceramic-based graft material might be an effective approach to promote osteogenesis in critical size bone defects. In this study, the domestic pig was used as an experimental model due to its anatomical, physiological, and metabolical similarities with humans as well as similar dentition to that of humans. Porcine TGSCs (pTGSCs) were isolated from mandibular third molar tooth germs of 6-month-old domestic pigs and characterized for their surface antigen profile. pTGSCs were sorted according to their STRO-1 expression as STRO-1(+) and STRO-1(-). In the first part of the thesis, in vitro proliferation and osteogenic differentiation capabilities of sorted cells were compared with each other to see the effects of STRO-1 sorting on their osteogenic capacities. STRO-1(+) cells exhibited a higher proliferation rate owing to their clonogenic properties. All three groups of cells were found differentiated into osteogenic lineage as shown by ALP activity, calcium deposition assay, detection of osteogenic mRNAs and proteins, and mineralization staining. According to differentiation analysis, STRO-1(+) cells did not show a better in vitro performance for osteogenesis compared to STRO-1(-) and US cells. In vitro results indicated that STRO-1(+) cells might require a heterogeneous population of cells including STRO-1(-) in their niche to perform their proposed role in osteogenesis. In the second part of the thesis, the osteogenic efficacy of transplanted allogenic pTGSCs in combination with ceramic and hydrogel based carrier systems on the healing of critical size defects in the pig calvaria was evaluated in vivo. MX10 hydrogel was found as an effective carrier for delivering stem cells and keeping them in the defect site. CM-DiI staining was useful for in vivo tracking of cells post transplantation.