**4.3 Using grafts, bioactive molecules, growth factors, and cement for bone regeneration**

#### *4.3.1 Autograft*

The gold standard and first bone replacements is autologous bone due to its high osteoinductive and osteoconductive properties [56]. The autografts contain natural

*Recent Advances, Challenges and Future Opportunities for the Use of 3D Bioprinting in Large… DOI: http://dx.doi.org/10.5772/intechopen.111495*

bone contents such as bone cells, growth factors, and natural extracellular matrix components such as collagen, non-collagenous proteins, and hydroxyapatite [56]. Although this method has good benefits, it also comes with challenges, for example, a secondary operation that causes suffering for the patient or limitations in the dimensions of the harvested bone, and fast absorption of autologous implant that should be regulated [57].

#### *4.3.2 Allograft*

Allografts are non-self-human that can be used as the other option for use in bone defects treatment and consist of one-third of all-bone grafts in North America [58]. The osteoinductivity and mechanical properties of the allograft can be reduced during the freezing, and sterilization processes, while the ECM can provide a natural scaffold for bone regeneration [59]. The main restriction of allografts is the risk of disease transmission (HIV, hepatitis B, hepatitis C, and other viruses), and the rareness of supplements [60].

#### *4.3.3 Bone morphogenetic proteins (BMPs)*

BMPs are the most popular subfamily of transforming growth factor beta that are necessary for fetal tissue development and fracture healing [61]. Bone morphogenetic protein-2 (BMP-2) shows high osteogenic potential and can be applied in bone regeneration [62]. It is the only osteoinductive growth factor with FDA approval for clinical usage, but its administration should be controlled. Excess administrated BMPs, especially in the first levels of bone mineralization can cause different side effects such as ectopic osteogenesis, osteolysis, bone cysts, local inflammation, traumatic injuries, postoperative fever, hemorrhage, or even cancers [63].

#### *4.3.4 Statins*

Statins, 3-hydroxy-3-methylglutaryl-coenzyme-A, are a family class of drugs reductase inhibitors, with impressive impact on the bone healing procedure. This can be due to the BMP-2 bone factor induction that can facilitate bone healing [64]. The most famous member of this group for bone regeneration applications is Simvastatin [65]. Simvastatin can accelerate bone regeneration by regulating BMP-2 gene expression and show an osteoinductive role through the Ras signaling pathway, and activate mitogen-activated protein kinases. Also, it can inhibit bone density reduction using estrogen receptor-alpha or BMP-2 induction [66, 67]. Feng et al. [68] demonstrated the capability of simvastatin to enhance osteoblast differentiation of mesenchymal stem cells in osteoporosis Sprague–Dawley rats. In another study, Shahrezaee et al. [69] investigated and compared the systemic delivery of atorvastatin, simvastatin, and lovastatin on bone healing. For this purpose, the drugs were orally administrated to the thirty ovariectomized female Sprague–Dawley rats, and the results were analyzed after 60 days. According to the achievements, atorvastatin had no significant effect on bone density, while simvastatin and lovastatin enhanced calcium levels, osteogenic gene expression, bone density, and biomechanical properties. The CT scan of different analyzed groups is shown in **Figure 1 (A-E)** and can prove the better function of simvastatin and lovastatin. In comparison, the simvastatin-treated sample showed a significantly higher resemblance to normal groups and demonstrated increased osteogenic genes and better biomechanical performance.

#### **Figure 1.**

*(A-E): Micro CT results of different analyzed groups after 60 days: (a): Normal, (B): Negative control, (C): Simvastatin, (D): Lovastatin, (E): Atorvastatin [69]. (F-K): The histological investigation of three control (F), sham (G), and experimental (H) groups after 1 week and control (I), sham (J), and experimental (K) groups after 8 weeks. The bottom images are the higher magnifications: (I1) fibrous connective tissue of control, (I2) control scaffolds covered with fibrous, (J1) fibrous connective tissue of sham, (J2) sham scaffolds covered with mixed tissue, (K1) cartilage and bone tissue of experimental group, (K2) experimental group covered with cartilage and bone tissue [70].*

### *4.3.5 Fibroblast growth factor-2*

Fibroblast growth factor-2 is a famous family member of fibroblast factors with unique properties such as mitotic promoters that can accelerate cell proliferation, collagen synthesis enhancer, angiogenesis, and osteogenesis [71]. However, its short half-life (3–10 min) should be regulated using different drug release carriers because excess Fibroblast growth factor-2 usage can cause side effects and depressant bone regeneration [72].

*Recent Advances, Challenges and Future Opportunities for the Use of 3D Bioprinting in Large… DOI: http://dx.doi.org/10.5772/intechopen.111495*

#### *4.3.6 Platelet-rich plasma*

Platelet-Rich Plasma (PRP) refers to the high concentration of platelets (two to five higher than blood) in a small volume of plasma with a lot of therapeutic applications due to the capability of platelets to secret different growth factors [73]. It was reported that approximately 300 bioactive cytokines exist in PRP and their release can trigger various cellular interactions and facilitate tissue regeneration. Although there is no international protocol for PRP usage [74]. The main growth factors are platelet-derived growth factor (PDGFaa, PDGFbb, PDGFab), TGFβ1/TGFβ2, VEGF, FGF, epithelial growth factor (EGF), bone morphogenetic protein (BMP), hepatocyte growth factor (HGF) and insulin-like growth factor (IGF) [75]. These growth factors can modulate inflammation, accelerate cell proliferation and differentiation, promote angiogenesis, and as a result accelerate bone regeneration [76]. The performance of PRP in large bone treatment is successful with a lower rate of pain score and shorter healing duration. However, a higher infection rate was reported [77].

#### *4.3.7 Insulin-like growth factors*

Insulin-like growth factor-1 (IGF-1) is one of the most abundant growth factors in bone matrix with a wide range of physiological functions [78]. The administration of IGF-1 lead to enhance osteoblasts growth and accelerate bone healing [79]. IGF receptor is a cell surface receptor named an Insulin-like growth factor 1 receptor and after binding to Insulin-like growth factors, it activates two main signaling pathways: the phosphoinositide 3-kinase (PI3 K)/AKT and the RAS/mitogen-activated protein kinase (MAPK) and leads to promoting cell proliferation, migration, and proliferation [80]. In addition to IGF-1, metformin can improve osteogenic differentiation and facilitate bone regeneration [81]. Shahrezaei et al. [82] reported that metformin coating of poly (lactic acid) and polycaprolactone scaffold increased the osteogenic and angiogenic markers expressions and accelerated the regeneration of rats' large bone defects.

#### *4.3.8 Bisphosphonates*

Bisphosphonates are drugs with inhibitory effects on the osteoclasts using increasing their apoptosis rate of them and interfering with resorption signals sent from the bone matrix [83]. These drugs are classified into two main categories with different mechanisms: nitrogen-containing bisphosphonates, and non-nitrogen-containing bisphosphonates. The non-nitrogen ones metabolized to analogs of adenosine triphosphate and interfere with mitochondrial activity and lead to cell death, while the nitrogen-containing ones inhibit farnesyl pyrophosphate synthase which can be effective in protein transportation and lead to cell death and inhibit osteoclast maturation [84].

#### *4.3.9 dECM*

The extracellular matrix (ECM) is a complex structure with various components including collagen, glycosaminoglycans, chondroitin sulfate, elastin, etc. [85]. In the decellularization procedure of ECM, the cells were eliminated with low harm to the natural structure and components of the ECM [85, 86]. Decellularized extracellular matrix (dECM) derived from natural ECM can be used as a biomaterial

in tissue engineering applications [87]. The dECM introduces a natural structure for bone replacement containing the bone components such as growth factors and cytokines [88]. Also, dECM can provide cellular activities that are necessary for bone healing [89].
