**4.4 Combining techniques**

Although each of the treatment methods mentioned above has benefits and potential effects, however, all methods face limitations. The simultaneous use and combination of the mentioned methods to provide a new treatment method can lead to the simultaneous use of the benefits of each method in such a way that they overcome each other's limitations. Leppik et al. [70] proposed a combined technique for the treatment of large bone defects by using tissue engineering and electrical stimulation. For this purpose, the test was analyzed *in-vitro* and *in-vivo*. The *in-vivo* experiment was performed on three different groups: large femur-defected rats treated with β -TCP scaffolds (control group), β-TCP scaffolds and Adipose tissuederived mesenchymal stem cells (AT-MSC) (sham group), electrical stimulation, β-TCP scaffolds, and Adipose tissue-derived mesenchymal stem cells (experimental group). The 3 cm defects were created in the right limbs of the rats under general anesthesia. After 1 week, Staining was performed using Alcian Blue, Orange-G, and Hematoxylin histological investigation was carried out (**Figure 1** (**F-K**)), the 80% defect gap was covered with soft tissue in the experimental group, while there was a high uncovered gap was seen in the control and sham groups. Although there can be a better condition in the sham group in comparison with control samples. After 8 weeks, histological observations showed that cartilage and bone tissue were formed properly in the experimental group.

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

In another combined treatment method, Qi et al. [78] used Electrical stimulation and insulin-like growth factor-1 (IGF-1) for bone regeneration. The effect of this method was tested on MC3T3-E1 cell behavior and the results were analyzed with different techniques. According to the achievements, the combination of electrical stimulation and IGF-1 acted synergistically and lead to higher expression of osteogenic marker genes and alkaline phosphatase activity, and cell mineralization. The results of alizarin red staining of the cell mineralization were shown in **Figure 2** (**A-H**). The combination techniques demonstrated higher calcium content in comparison with the cells that only receive the IGF-1 factor. On the other hand, when the concentration of IGF-1 was 100 and 200 ng/ml a higher calcium content was detected. In another clinical trial, Combal et al. [102] combined the Masquelet-Induced Membrane and Capanna Vascularized Fibula with an Allograft large femoral bone defects and introduced it as Capasquelet. The technique can be used for the treatment of bone defects with at least 10 cm loss. For this purpose, four patients suffering from tumors/traumatic bone defects were operated on with this combination technique. The result proved the important effect of the induced membrane in the regeneration process with fast allograft and fibula union and early weight-bearing **Figure 2** (**I-J**).

#### **4.5 Novel techniques**

#### *4.5.1 Cell therapy*

Few cell types can form heterotrophic bones while many cell types can create mineralized tissues [103]. Progenitor cells are the best cell population for cell therapy applications. The number of the needed cells depends on different factors such as bone fracture types and size, cell source, therapeutic method, and biomaterials, whereas usually for a 4 cm large bone defect approximately 600 million cells are sufficient [104]. Mesenchymal stem cells are extensively studied in bone regeneration with favorable clinical application, especially in dental and orthopedic fields [105]. Furthermore, MSCs can regulate physical contact between cells and enhance cell–cell interactions for osteogenesis [106]. Although these cells can be isolated from different parts of the body, the gold standard is included bone marrow-derived MSCs (BM-MSCs) due to their easiness and high osteogenic capacity [107]. BMSCs are the most popular cell population for bone treatment over 50 years ago [104]. In the Hernigou process, the bone marrow aspirates to the bone defect sites [106]. Furthermore, the infusion of BMSCs is used for the treatment of children with severe osteogenesis imperfect [108].

#### *4.5.2 Tissue engineering*

The limitations of the previous bone replacements such as donor-site scarcity, high risk of disease transmission, and immune rejection made the scientists find a new solution. Tissue engineering introduced promising strategies for bone regeneration [109–111].

Tissue engineering consisted of cells, scaffolds, and bioactive molecules. Both natural and synthetic materials can be used for scaffold fabrication in tissue engineering with their unique properties. Natural materials provide better biological recognition and cellular interactions [112]. Synthetic materials can be modified according to the requirements and the source is usually abundant. On the other hand, the material

#### **Figure 2.**

*(A-H): Alizarin red staining results of the calcium content after 21 days of electrical stimulation and IGF-1 factor treatment (a). The samples are (B) control, (C) IGF (50 ng/ml), (D) IGF (100 ng/ml), (E) IGF (200 ng/ml), (F) ES + IGF (50 ng/ml), (G) ES + IGF (100 ng/ml), and (H) ES + IGF (200 ng/ml) [78]. (I-J): Control X-rays for patient No. 1 (I) 3 months (J) 10 months postoperatively [102].*

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

should be modified to overcome usual synthetic materials disadvantages such as low material-tissue integrity, low vascularization, poor cell attachment, and therefore functional bone regeneration [113, 114].
