Interestingly, human bone tissue has a relatively high potential for regeneration (Bessa et al., 2008a; Reddi, 2005); however, the natural healing capacity of bone tissue may fail if the bone defect is large, also referred to as critical-sized. Bone autografts are considered a gold standard for bone reconstruction due to their histocompatibility and non-immunogenicity and possess the essential factors for bone healing: osteoinductive factors such as bone morphogenetic proteins, osteoprogenitor cells required for osteogenesis, and functional material for osteoconduction such as three-dimensional and porous matrix structure (Amini et al., 2012). Nevertheless, the use of autografts requires an additional surgical operation for bone harvesting, and several studies have reported significant
limitations and complications such as donor site injury and morbidity, deformity, scarring and surgical risks (Amini et al., 2012). Bone tissue engineering is a novel multidisciplinary approach for bone regeneration and requires the collaborative work of biologists, engineers, and surgeons (O'Keefe and Mao, 2011). Several critical factors exist in bone tissue engineering that affect the final outcome of regenerated bone. A biocompatible scaffold is a key component that should provide a supportive structure for bone regeneration. The design of biocompatible scaffolds requires a balance of an osteoinductive cellular microenvironment, diffusion of soluble factors, sufficient flexibility, and mechanical loading appropriate for the anatomical site (Mravic et al., 2014). In certain cases, osteogenic cells may be seeded on a biomaterial scaffold in combination with growth signals to direct osteogenic differentiation.
Moreover, sufficient vascularization is essential for the survival of newly formed bone to provide nutrients for the growing tissue (Weigand et al., 2015).
2.7.1 Biomaterials in clinical bone applications
A biomaterial is defined as a nonviable material used in a medical device that is intended to interact with biological systems (Williams, 1999). Biocompatibility is essential for biomaterials and is defined as the ability of a material to perform with an appropriate host response in a specific situation (Williams, 1999). The first materials referred as biomaterials were invented almost 50 years ago for the purpose of simply replacement of diseased, damaged or aged tissues (Hench et al., 2004).
Thus, these materials were selected to match the physical properties of the replaced tissues with a minimal toxic response in the host and thus were as bioinert as possible (Hench et al., 2004). In the 1970s, Hench et al. discovered that certain bioglasses containing CaO and SiO2 were able to form a strong adhesive bond with bone and soft connective tissues (Hench, 1998). These biomaterials became known as bioactive materials due to a controlled reaction in the physiological environment.
Subsequently, bioactive glass (BAG) and beta-tricalcium phosphate (β-TCP) ceramic have been widely studied and used for bone tissue engineering purposes due to their biocompatibility and ability to support osteoblastic growth and maturation (Baino and Vitale-Brovarone, 2011; Q. Z. Chen et al., 2006; Haimi et al., 2009a;
Mesimaki et al., 2009; Sandor et al., 2013; Thesleff et al., 2011; Vitale-Brovarone et al., 2007; Yuan et al., 2001). The BAG develops a carbonated phosphate surface layer that allows chemical bonding to the host bone. This bone-bonding behavior is referred to as bioactivity and has been associated with the formation of a carbonated
hydroxyapatite (HCA) layer on the glass surface after implantation (Hench, 1998). A biologically active HCA layer has been shown to be essential for the bone bonding.
In addition to BAGs, hydroxyapatite (HA) and related calcium phosphates have shown excellent bioactive properties and abilities in bone bonding. Approximately 60 wt% of bone consists of HA Ca10(PO4)6(OH)2, and therefore, HA and related calcium phosphates such as α-TCP and β-TCP have been investigated for use as scaffold materials in bone tissue engineering, as previously reviewed (Rezwan et al., 2006). Due to the high similarity of the chemical and crystal structure of calcium phosphates to bone minerals, these materials exhibit a superior biocompatibility with bone tissue. Although they may not be osteoinductive, calcium phosphates have osteoconductive abilities and bind directly to bone under certain conditions (Rezwan et al., 2006). Osteoinduction is defined as the stimulation and activation of host stem cells from the surrounding tissue or, alternatively, activation of transplanted stem cells for differentiation into bone-forming osteoblasts, also in ectopic tissues. In contrast, osteoconduction is defined as the ability of a material to serve as a scaffold to which bone cells attach, migrate and divide, but osteogenesis is not directly induced (Albrektsson and Johansson, 2001). With advanced material processing techniques, optimal porous and interconnected 3D structures can be manufactured to further support osteoblastic growth and maturation (Rezwan et al., 2006).
2.7.2 Bone morphogenetic proteins
To identify more efficient and reliable methods for osteogenic differentiation of MSCs, several growth factors and cytokines have been investigated for their potential to direct MSC osteogenesis. When osteoinductive biomaterials are combined with optimal osteoinductive factors, effective osteogenic differentiation can be achieved.
The BMPs were discovered in 1965 and are probably the most important growth factors in bone formation and healing (Bessa et al., 2008a; Reddi, 2005). In 1972, it was suggested by Reddi and Huggins that BMPs are responsible for the initiation of a cascade of developmental events in which progenitor cells are induced to produce bone cells, thus leading to bone regeneration (Bessa et al., 2008a). The BMPs belong to the transforming growth factor-β (TGF-β) superfamily that includes several other growth factors, such as activins, inhibins and TGF-βs. These growth factors have functions in several tissues and organs, but BMPs play significant roles, especially in bone and cartilage formation (Bessa et al., 2008a). BMP superfamily includes a large number of growth factors from BMP-2 to BMP-18 of which only BMPs -2, -4, -6, -
7, and -9 are known to induce complete bone morphogenesis. In contrast, BMP-3 and -3b have been shown to have negative effect on osteogenesis by downregulating the expression of ALP in bone cells (Hino et al., 2004). Currently, human BMPs can be produced in large amounts using recombinant technology, and in 2002, the FDA approved two models of collagen sponges (Infuse™, Medtronik, US/Wyeth, UK;
Osigraft™, Stryker Biotech) that deliver recombinant human BMP-2 or BMP-7 for human use as an alternative to bone grafts for spinal fusion and long bone fractures (Bessa et al., 2008b).
Signaling of BMP is mediated to the cell nucleus through serine-threonine kinase receptors on the cell surface, where a specific intracellular pathway is activated, leading to gene transcription and finally affecting the cell proliferation and differentiation (Shi and Massague, 2003). Smads are the main signal transducers of these complex pathways that are strictly regulated (Bessa et al., 2008a; Derynck and Zhang, 2003). Upon BMP binding, type I receptors phosphorylate receptor- regulated Smads (R-Smads) that form a heterotrimeric complex with one Co-Smad, which translocates the signal into the nucleus and modulates gene transcription in co-operation with other transcription factors. Smads regulate the transcription of several genes such as RUNX-1, -2 and -3, OSX, HOXC-8 and MyoD (Bessa et al., 2008a). One of the most studied early markers of osteogenic differentiation is RUNX-2, the expression of which is low in mesenchymal cells and is further induced after BMP signaling (Ito and Miyazono, 2003). RUNX-2 regulates processes such as bone formation and hematopoiesis and upregulates several osteogenic markers such as ALP, osteocalcin and osteopontin (Bessa et al., 2008a).
2.7.3 Towards allogeneic bone treatments with ASCs
For practical purposes, a cell product should be available as an off-the-shelf product, immediately upon demand at the point of care (Liu et al., 2013). The current use of autologous ASCs in a clinical setting does not meet this criterion. Isolation and expansion of ASCs is time-consuming and importantly, cells derived from aged donors or cancer patients should be replaced, possibly by the allogeneic cells of young healthy donors (Gu et al., 2014). Therefore, the ability to use allogeneic ASCs for bone repair is more suitable for clinical demands. Gu et al. have observed that similar to undifferentiated ASCs, osteogenically differentiated ASCs show low expression for MHC I antigens and are negative for MHC II (Gu et al., 2014) and concluded that the allogeneic differentiated ASCs maintain their low
immunogenicity and can be used in allogeneic settings, as demonstrated in an ulnar bone rabbit model without any immunosuppressive therapies. The use of allogeneic osteogenically differentiated ASCs for the treatment of critical-sized bone defects in vivo was also investigated by Liu et al. (Liu et al., 2013), who demonstrated the potential of allogeneic ASCs in the treatment of bone diseases using an immunocompetent canine cranial model without immunosuppressive therapies. As previous studies have demonstrated, ASCs show low immunogenicity and possess immunomodulatory functions both in vitro and in vivo (Section 2.6.1), but translation of these findings to the clinical settings is still challenging. Limitations remain, such as differences in immune systems between human and animal species associated with many of the used models, which may confuse interpretation of the results if the aim is to provide safe, effective, and reproducible treatments for the patient. Well-designed and standardized clinical trials are necessary to verify the safety and efficacy of ASCs for allogeneic stem cell treatments and for immune modulating therapies. Clinical trials for ASCs are described in more detail in Section 2.9.