**2. Stem cell sources**

Over the last few decades, stem cells have emerged as a key player in tissue engineering, both for in vitro generation of bones, and in vivo bone regeneration. Two hypotheses have been proposed to describe the mechanism of healing affected by stem cells. In the conventional approach, osteogenic stem/progenitor cells are proposed to participate in new bone through direct differentiation into functional tissue. More recently, it has emerged that trophic factors secreted by administered stem/progenitor cells can promote functional tissue regeneration [19]. Much research is currently focused on understanding the influence of stem cell origin and culture conditions on clinical outcome.

#### **2.1. Adult stem cell sources**

The human body retains a small amount of osteoprogenitor cell population with differentiation potential even in adulthood. The main osteogenic progenitor cells – mesenchymal stem cells (MSCs) are considered to consist of a variety of cell populations, with no unique marker to delineate a MSC to-date [20]. Therefore, in some studies, the term "mesenchymal stem cells" is used to represent osteoprogenitor cells [21]. The current consensus on the characteristics of a bona-fide MSC should include: 1) expression of cell-surface markers which are non-haemo‐ poietic, non-endothelial and incorporates a number of other cell-surface markers, including CD73, CD105 and CD90, and 2) the capacity for tri-lineage differentiation into osteocytes, chondrocytes and adipocytes [22].Common sources of MSCs include bone marrow, fat tissue and the periosteum [23].

MSCs are most abundantly found in the bone marrow (BM), and BM-MSCs are widely utilised for both autologous or allogeneic transplantations [20]. BM-MSCs can be easily expanded in culture, and induced to differentiate into osteoblasts through culture in a high phosphate environment [24]. In tissue engineering applications, BMMSCs have been used to generate bone graft by loading into appropriate scaffolds, followed by osteogenic culture [25],. BM-MSCs may offer favourable characteristics, including availability as an autologous cell source, and thus, non-immunogenic cell source [26]. In contrast to suggestions that BM-MSC are immunoprivilleged and thus suitable for allogeneic – unmatched transplantation [27], a few studies have emerged that reported rejection of murine BM-MSCs following infusion into immune competent recipients [28]. Besides, adult BM-MSCs though advantageous in autolo‐ gous source availability, have shown relatively low efficiency in osteogenic differentiation in vivo in several comparative studies [29, 30].

The periosteum is involved in extraosseous fracture repair, and is thus also thought to contain osteoprogenitors. Indeed, stem cells derived from periosteal tissue showed better mineraliza‐ tion and neovascularization ability than adult bone marrow derived cells and demonstrating good efficacy in healing a calvarial defect model [31]. However, its potential as a cell source is limited by the low volume of periosteum which can be harvested, as well as the complexity of the harvesting process [32]. Further optimization in terms of isolation and culture conditions will be required before it can be a practical source.

Adipose tissue represents another potential source of osteogenic stem cells [33]. Adipose derived stem cells (ADSCs) can be isolated from fat tissues harvested by liposuction or surgical fat section, which are typically considered medical waste [34]. Similar to BM-MSCs, ADSCs respond to osteogenic induction through upregulation of alkaline phosphatase and other key osteogenic proteins, and differentiate into osteoblasts capable of depositing minerals [35]. Additionally, ADSCs have been shown to ne efficacious in bone repair, facilitating the repair of critical size defects in both calvarial and femoral segmental defects [36, 37]. However, the efficacy of bone repair by using ADSCs alone is relatively low compared with marrow-origin MSCs and more primitive MSCs [38, 39].

Theadult stemcells representarichautologous source forbone tissue engineering,andthe longhistorystudyaccumulatematuremethodologytofacilitate their applicationinpre-clinicaltrials or even clinical trial [40, 41]. Yet the low efficiency of in vitro bone generation and in vivo bone regenerative capacity may call for optimized induction strategy or alternative sources.

#### **2.2. Embryonic Stem Cell (ESC) and induced Pluripotent Stem Cell (iPSC)**

In the last decade, the osteogenic induction of embryonic stem cells (ESCs) and the creation of induced pluripotent stem cells (iPSCs) presented new cell sources for bone tissue engineering [42, 43]. However, expansion of ESCs and iPSCs to clinically useful numbers is logistically challenging, and autologous use is not possible in the case of embryonic stem cells. Moreover, precise control of differentiation is necessary before applying to clinical use. This is especially so since the presence of undifferentiated "rogue cells" may result in tumour formation after transplantation [23].

ESCs are defined by their pluripotency, and can be induced into osteogenic cells through different methods in vitro [44, 45]. It is showed that human ESCs can be induced into osteogenic cells and form mineralized bone in vivo without presence of teratoma [46]. However, more investigations will be required to address the potential of teratoma in embryonic stem cells transplantation. Mature methods for specific osteogenic differentia‐ tion are also needed to overcome non-specific differentiation before clinical implementa‐ tion can be contemplated.

As an alternative, iPSCs have recently emerged as a potential cell source for regenerative medicine [43]. Compared to ESCs, iPSCs face fewer ethical challenges, and are able to serve as an autologous cell source. iPSCs have been successfully differentiated into osteoblasts like cells [47], or through induction into a MSC phenotype [48, 49], allowing ready expansion of the iPSC-derived MSC before their direct differentiation into osteoblasts. However, similar issues to that of ESC remain, and concerns remain over the potency of the cells. Additionally, the concept of reprogramming remains poorly understood, and leaving questions on the fate of cells in vivo [49]. Therefore, although the differentiation capacity qualifies the iPSC as a potential source for bone engineering, yet a lot more efforts have to be done before it can be competent.

#### **2.3. Fetal stem cell sources**

delineate a MSC to-date [20]. Therefore, in some studies, the term "mesenchymal stem cells" is used to represent osteoprogenitor cells [21]. The current consensus on the characteristics of a bona-fide MSC should include: 1) expression of cell-surface markers which are non-haemo‐ poietic, non-endothelial and incorporates a number of other cell-surface markers, including CD73, CD105 and CD90, and 2) the capacity for tri-lineage differentiation into osteocytes, chondrocytes and adipocytes [22].Common sources of MSCs include bone marrow, fat tissue

MSCs are most abundantly found in the bone marrow (BM), and BM-MSCs are widely utilised for both autologous or allogeneic transplantations [20]. BM-MSCs can be easily expanded in culture, and induced to differentiate into osteoblasts through culture in a high phosphate environment [24]. In tissue engineering applications, BMMSCs have been used to generate bone graft by loading into appropriate scaffolds, followed by osteogenic culture [25],. BM-MSCs may offer favourable characteristics, including availability as an autologous cell source, and thus, non-immunogenic cell source [26]. In contrast to suggestions that BM-MSC are immunoprivilleged and thus suitable for allogeneic – unmatched transplantation [27], a few studies have emerged that reported rejection of murine BM-MSCs following infusion into immune competent recipients [28]. Besides, adult BM-MSCs though advantageous in autolo‐ gous source availability, have shown relatively low efficiency in osteogenic differentiation in

The periosteum is involved in extraosseous fracture repair, and is thus also thought to contain osteoprogenitors. Indeed, stem cells derived from periosteal tissue showed better mineraliza‐ tion and neovascularization ability than adult bone marrow derived cells and demonstrating good efficacy in healing a calvarial defect model [31]. However, its potential as a cell source is limited by the low volume of periosteum which can be harvested, as well as the complexity of the harvesting process [32]. Further optimization in terms of isolation and culture conditions

Adipose tissue represents another potential source of osteogenic stem cells [33]. Adipose derived stem cells (ADSCs) can be isolated from fat tissues harvested by liposuction or surgical fat section, which are typically considered medical waste [34]. Similar to BM-MSCs, ADSCs respond to osteogenic induction through upregulation of alkaline phosphatase and other key osteogenic proteins, and differentiate into osteoblasts capable of depositing minerals [35]. Additionally, ADSCs have been shown to ne efficacious in bone repair, facilitating the repair of critical size defects in both calvarial and femoral segmental defects [36, 37]. However, the efficacy of bone repair by using ADSCs alone is relatively low compared with marrow-origin

Theadult stemcells representarichautologous source forbone tissue engineering,andthe longhistorystudyaccumulatematuremethodologytofacilitate their applicationinpre-clinicaltrials or even clinical trial [40, 41]. Yet the low efficiency of in vitro bone generation and in vivo bone

regenerative capacity may call for optimized induction strategy or alternative sources.

and the periosteum [23].

602 Regenerative Medicine and Tissue Engineering

vivo in several comparative studies [29, 30].

will be required before it can be a practical source.

MSCs and more primitive MSCs [38, 39].

Compared with adult stem cells, fetal stem cells are relatively more primitive, and have higher proliferative and differentiation capacity. MSCs have been derived from various fetal tissues in early to mid-gestation, including the blood, bone marrow, liver, pancreas, kidney thymus and bone [50]. Of these, fetal BM-MSC has shown particular utility for bone tissue engineering as reviewed extensively by Zhang et al [19]. However, its availability may be limited by ethical issues especially where fetal tissues are used to derive MSC [51].

As an alternative source, MSC has also been derived from umbilical cord blood at term gestation, albeit at very low frequencies [51], with optimised protocols achieving up to 60% success in its derivation [52]. Aside from the cord blood, MSC has also been derived from the umbilical cord vessels and matrix, the placenta and fetal membranes [53]. Although these sources of MSC are plentiful and readily harvested, their utility for bone tissue engineering is somewhat limited compared to fetal bone marrow derived MSC [19, 29].

#### **2.4. Conclusion**

Stem cell sources for bone tissue engineering have been widely explored recently, and several studies have been conducted to compare the different cell sources [21, 29, 30, 44, 54]. Table 1 below summarizes some of these studies and compares the main properties of different stem cell sources.


**Table 1.** A comparison of stem cell sources based on comparative studies
