**13. Cell therapy**

applied in combination with other promoting factors like BMP and PDGF to improve it's the regenerative features [65-67]. Despite all the important roles of VEGF investigated and presented in the literature most studies showed that this growth factor is less inductive than

**Basic fibroblast growth factor (bFGF).** bFGF is an important growth factor in wound healing, formation of granulation tissue and remodeling [68]. Several studies evaluated the effect of bFGF in bone regeneration; however its role is not as important as other factors like BMP [55].

**Transforming growth factor beta (TGF-β).** TGF-β is a group of proteins released from several tissues including macrophages and plays an important role in healing. The bone regenerative features of rhTGF- β1, rhTGF-β2, and TGF-β3 have been evaluated in different investigations. The usual carrier for the delivery of this growth factor in these studies is a gelatinous matrix. Some of these researches have shown the positive influence of this growth factor in bone

**Indications.** The most common usage of growth factors is in implant surgery. The defects created during the procedure or post-operative bone dehiscences may be corrected with the application of growth factors. **Advantages.** Growth factors are presented as an alternative for bone grafts in reconstruction of maxillofacial defects. These proteins reduce the morbidity of the patients by removing the need of harvesting bone grafts. These factors are responsible for the major events in regeneration including angiogenesis, cell differentiation, mitogenesis, and bone formation [69]. Furthermore the combination of these proteins with bone grafts promotes

**Disadvantages.** The high costs of producing growth factors are the major limitations for using these materials in humans. Production of recombinant growth factors as rhBMP and rhPDGF requires a period of time and high costs [70]. Application of growth factures is very technique sensitive and the clinician should be an expert in this procedure. Choosing a slow releasing scaffold is still a challenge among surgeons to use with the growth factor as a carrier. The appropriate dosage and useful concentration of these proteins in bone regeneration is another controversial issue which should be resolved. The excess amount of growth factor or wrong application of them may lead to ectopic bone formation and result in insufficient correction of

Biomaterial carriers are needed for delivery and sustained release of growth factors. The application of growth factors without a proper carrier is very hard and their handling is almost impossible. There is no universal carrier for this purpose. Several biomaterial carriers have been suggested to be effective in delivery of certain growth factors and accelerate bone formation. The osteoconductive ability of the scaffold should be considered in choosing the right carrier for the purpose. The advantages and disadvantages of usual growth factor carriers

the generation of new bone and facilitates healing of the defects.

BMP in bone regeneration [55.[

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regeneration [55].

the deficiencies.

are presented in Table 1.

**12. Carriers in bone regeneration**

#### **13.1. MSCs harvesting sources**

Cell therapy is a new technique in reconstruction of bone deficiencies presented as an alter‐ native for bone grafting. The self-renewal ability and the capability of differentiating to osteogenic cells have made the stem cells a popular source in regeneration of bone defects. Several tissues have been suggested as the source of stem cells including fat, umbilical cord blood, lung, liver, skin, periosteum, and skeletal muscle [71]. Recently dental pulp was used as a new origin for extracting stem cells for regenerative purposes [72]. The usual source of MSCs in each study and various models is different. According to the literature the most common origin to harvest the MSCs in rat models is human bone marrow-derived mesenchy‐ mal stem cells (hBMSCs) usually extracted from femur or tibia [73-75]. The most common source in harvesting MSCs to regenerate the bone defects in rabbit and dog models as well as human studies is the iliac bone [71]. By considering the reduced differentiation potential of MSCs harvested from bone marrow investigators have attempted to find new sources of MSCs. Birth associated tissues like umbilical cord and dental pulp as well as adipose tissue are new sources that have been found to contain MSCs [76].

## **13.2. MSCs culture and differentiation protocol**

MSCs as a compartment of various cell populations are aspirated from the selected origin like the iliac crest or buccal fat pad. The aspirated cells are cultured in a medium with Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) for 3 h in a 37 degrees 5% CO2 incubator. Then the non-adherent cells are discarded after three hours and adherent cells are washed with phosphate-buffered saline (PBS) and fresh medium is replaced. The culture is treated with 0.5 ml of 0.25% trypsin containing 0.02% ethylene-diamine-tetra-acetic acid (EDTA) for 2 min at room temperature when the primary culture is confluent. A purified population of MSCs can be obtained 3 weeks after the initiation of culture [77]. The third generation of the cells is usually used in the studies (Figure 20) [78, 79]

**Figure 20.** A, Proliferation of MSCs under light microscopy. B, Alizarin red staining for evaluating differentiation of MSCs to osteoprogenitor cells. Mineralization of the extracellular matrix is visualized by this staining technique. C, Oil red staining of MSCs, depicted adipogenic differentiation.

#### **13.3. MSCs culture on scaffolds**

Several investigations have evaluated the efficacy of stem cell regenerative ability on animals [78-82]. The stem cells should be implanted on an appropriate scaffold before delivery to the surgical site. According to the literature TCP is an efficient carrier for the stem cells to be loaded on and transplanted to the surgical site [71, 80, 81]. After preparation the choice carrier for reconstruction purpose it should be immerged into the medium impregnated with the MSCs. The MSCs should be implanted on the scaffold after 2 hours in 37ºC. Scanning electron microscope (SEM) is a useful assay to evaluate the presence of MSCs on the scaffold (Figure 21). Tripoding adherence of MSCs on the scaffold can be assessed under SEM [78].

#### **13.4. Current trends in MSCs application in bone regeneration**

Presentation MSCs as a novel regenerative technique in reconstructing bone defects provoked lots of investigators to evaluate the efficacy of MSCs application in oral and maxillofacial areas. Omitting the need for bone harvesting from a donor site and reducing the patient morbidity by application of MSCs in bone reconstruction promises a bright future for researchers around the world. Comparing the application of MSCs in bone regeneration to the control groups which bone materials were used has shown the increase of new bone formation. Implantation of MSCs together with bone minerals improves the regeneration of bone defects by delivery

**13.2. MSCs culture and differentiation protocol**

538 A Textbook of Advanced Oral and Maxillofacial Surgery Volume 2

red staining of MSCs, depicted adipogenic differentiation.

**13.3. MSCs culture on scaffolds**

MSCs as a compartment of various cell populations are aspirated from the selected origin like the iliac crest or buccal fat pad. The aspirated cells are cultured in a medium with Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) for 3 h in a 37 degrees 5% CO2 incubator. Then the non-adherent cells are discarded after three hours and adherent cells are washed with phosphate-buffered saline (PBS) and fresh medium is replaced. The culture is treated with 0.5 ml of 0.25% trypsin containing 0.02% ethylene-diamine-tetra-acetic acid (EDTA) for 2 min at room temperature when the primary culture is confluent. A purified population of MSCs can be obtained 3 weeks after the initiation of culture [77]. The third

**Figure 20.** A, Proliferation of MSCs under light microscopy. B, Alizarin red staining for evaluating differentiation of MSCs to osteoprogenitor cells. Mineralization of the extracellular matrix is visualized by this staining technique. C, Oil

Several investigations have evaluated the efficacy of stem cell regenerative ability on animals [78-82]. The stem cells should be implanted on an appropriate scaffold before delivery to the surgical site. According to the literature TCP is an efficient carrier for the stem cells to be loaded on and transplanted to the surgical site [71, 80, 81]. After preparation the choice carrier for reconstruction purpose it should be immerged into the medium impregnated with the MSCs. The MSCs should be implanted on the scaffold after 2 hours in 37ºC. Scanning electron microscope (SEM) is a useful assay to evaluate the presence of MSCs on the scaffold (Figure

Presentation MSCs as a novel regenerative technique in reconstructing bone defects provoked lots of investigators to evaluate the efficacy of MSCs application in oral and maxillofacial areas. Omitting the need for bone harvesting from a donor site and reducing the patient morbidity by application of MSCs in bone reconstruction promises a bright future for researchers around the world. Comparing the application of MSCs in bone regeneration to the control groups which bone materials were used has shown the increase of new bone formation. Implantation of MSCs together with bone minerals improves the regeneration of bone defects by delivery

21). Tripoding adherence of MSCs on the scaffold can be assessed under SEM [78].

**13.4. Current trends in MSCs application in bone regeneration**

generation of the cells is usually used in the studies (Figure 20) [78, 79]

**Figure 21.** SEM Evaluation of MSCs (×50). SEM analysis shows lodging of the cells within the pores of the scaffold.

of the cells responsible for synthesizing new bone directly to the defect site [80]. Experimental studies on rat models have shown that the maximum bone formation was 2.53 mm in the β-TCP/MSC group 6 weeks after the surgery [79]. Histomorphometric analysis of the rabbit experiments at 6 and 12 weeks post-operation has demonstrated significantly higher bone formation in the group which MSCs were applied in combination with PRGF and nano-HA [78]. Histological analysis of rabbit models in other investigations demonstrated that the mean amount of vertical bone was higher in the MSCs group than the control group (2.09 mm versus 1.03 mm) after two months [82]. Choosing the appropriate scaffold for delivery of MSCs is important to gain the highest rate of new bone formation. The different studies on dog mandibles have indicated the importance of scaffolds on bone formation [61, 80, 81]. Jafarian et al. showed that six weeks after delivering dog BMSCs with biphasic scaffold (HA/TCP) or NBBM (Bio-Oss) in a through-and-through 10-mm mandibular defect, new bone formation was 65.78% and 50.31%, respectively [80]. Histomorphometric analysis in Khojasteh et al. study showed that after 8 weeks of the scaffold implantation (polycaprolactone-tricalcium phosphate (PCL-TCP)) higher amount of lamellar bone was generated more on the test side (48.63%) than control side (17.27%) [81]. Khojasteh et al. in another study applied MSCs with recombinant platelet derived growth factor (rh-PDGF) in mandibular defects in dogs; however the result showed only 21.52% new bone formation [61].

Nowadays the major concern about the application of MSCs in bone defect reconstruction is its effectiveness and delivery technique in human cases. Application of MSCs in sinus floor lifting in posterior atrophic maxilla has been assessed in human trials and reports. Several organic and inorganic materials have been suggested for sinus augmentation in the literature. MSCs seeded on an appropriate scaffold are new regenerative techniques advocated for this procedure. High mean percentage of new generated bone in these studies may indicate the important inductive potential of MSCs [83]. Alveolar cleft of maxilla is another recipient site for applying MSCs instead of autografts to reduce morbidity. Some authors have shown successful results of using MSCs in alveolar clefts [84] whilst some others did not [85]. The amount of new bone formation may be insufficient for reconstruction of clefts; however it is usually enough for orthodontic tooth movements [85]. The combination of MSCs and a growth factor may increase their inductive and regenerative potential; however the results were not satisfactory yet [86].

**Indications.** Alveolar clefts are examples of the maxillofacial defects which cell therapy may be useful [85, 86]. Cell therapy is also indicated in augmentation of the sinus floor [83].

**Advantages.** It avoids the drawbacks of bone grafting like donor site morbidity. The stem cells are able to differentiate to different cell linings based on the combined growth factor. By extracting the cells from the own patient autologous transplantation is possible and no immune-suppressive therapy is necessary.

**Disadvantages.** Accessibility and the requirement for a large amount of cells are the main disadvantages of cell therapy as well as expenditure of time and money to provide the adequate cells for regeneration in large defects. The genetic damage occurrence of adult stem cells is a possibility in old patients. Embryonic stem cells have the risk of rejection and uncontrolled proliferation (turning into a teratoma).
