The modification by bone-specific genes enables MSCs to secrete certain growth factors, cytokines, or hormones and to change intracellular transcription level by regulating the expression of transcription factors and transcription coactivators (Table 1). The engineered MSCs are then expanded in vitro and used for BTE.
Many members of the whole bone morphogenetic proteins (BMPs) superfamily correlate with bone, cartilage, and joint development (Table 2). BMP-2, approved by Food and Drug Administration (FDA) for clinical practice, is the most potent member in promoting bone and cartilage development and therefore wins a popular choice for MSCs-based BTE (Lieberman et al.,1998; Lieberman et al.,1999; Lou et al.,1999; Turgeman et al.,2001; Olmsted-Davis et al.,2002; Blum et al.,2003; Gugala et al.,2003; Park et al.,2003; Riew et al.,2003; Tsuda et al.,2003; Kumar et al.,2004; Hasharoni et al.,2005; Egermann et al.,2006; Feeley et al.,2006). The BMP-2-modified MSCs are proven to increase the alkaline phosphatase (ALP) activity, mineralization, and cell proliferation in vitro and induce ectopic bone formation, heal critical size bone defect, repair fracture, and trigger spinal fusion in vivo (Lou et al.,1999; Moutsatsos et al.,2001; Turgeman et al.,2001; Blum et al.,2003; Park et al.,2003; Riew et al.,2003; Tsuda et al.,2003; Hasharoni et al.,2005; Egermann et al.,2006; Feeley et al.,2006). BMP-7 plays a key role in osteoblast differentiation, and there is only one study in which MSCs are engineered with BMP-7. Engineered MSCs are seeded in the distraction gaps of the mandibles, and the study demonstrates accelerated callus formation in distraction osteogenesis (Hu et al.,2007). BMP-4 is associated with bone and cartilage development and fracture repair. BMP-4-modified MSCs increase trabecular bone mineral density (BMD) and can heal critical sized femur defects in adult rats (Rose et al.,2003; Zhang et al.,2004). BMP-6 is characterized by maintaining joint integrity in adults. Interestingly, BMP-6-modified bone marrow MSCs (BM-MSCs) demonstrate accelerated osteogenic differentiation and mineralization in vitro, but the result is less robust than BMP-2-modified BM-MSCs (Zachos et al.,2006). BMP-9 only has a moderate osteoinductive capability (Celeste et al.,1990). However, BMP-9-engineered MSCs stimulate ALP activity in vitro and induce ectopic bone formation in vivo (Ploemacher et al.,1999; Helm et al.,2000; Varady et al.,2001; Dumont et al.,2002; Dayoub et al.,2003; Li et al.,2003), promoting its future use in BTE.
The genes encoding systemic hormone may not regularly be considered as candidates for BTE because the main biological function of the hormone will only be reached while distributed systemically. However, when the gene of human growth factor (hGH) is engineered into dog and mouse MSCs in vitro, and reintroduced into the foreleg vein, the iliac crest marrow, and the femur marrow, higher level of hGH is detected in the blood of the recipient animal a few days later (Hurwitz et al.,1997; Suzuki et al.,2000). In this regard, BM-MSCs may be used for the delivery of GH or other systemic hormones targeting at bone to treat systemic bone disorders such as osteoporosis.
The genes of transcription factors accounting for osteoblast differentiation of MSCs are also on the list. Overexpression of Runx2 and osterix in engineered MSCs may direct osteoblastic lineage differentiation and suppress other lineage differentiation (Merriman et al.,1995; Nakashima et al.,2002). Runx2-engineered MSCs directly enhance the repair of critical-sized calvarial defects when implanted solely (Zheng et al.,2004; Zhao et al.,2007) and form more bone when used in BTE (Zhao et al.,2005; Byers et al.,2006). Despite this, Runx2 and BMP2 also produce a strong synergistic effect after both are engineered into MSCs (Yang et al.,2003). Osterix may act downstream of Runx2 (Akiyama et al.,2005), and neither endochondral nor intramembranous bone formation occurs in osterix-null mutant mice (Nakashima et al.,2002). In one study, osterix-engineered BM-MSCs in vivo form five times the amount of new bone than control in healing of the calvarial bone defects in mouse (Tu et al.,2007).