**4. Quantity and quality of T cells, TIL, and CAR-T used in acknowledged clinical trials**

easy-to-handle closed system as ATMP in clinical settings near to patients [44, 45]. A particular advantage is that mass amounts of individual NK cells can be produced in a relatively inexpensive way due to low costs for selection, medium, activation, compared with other functionalized immune cells. NK cells can be expanded 2000–50,000-fold in designated perfusion bioreactors, whereas that has by far not been achieved in culture flasks. Adjuvant treatment of stem cell-transplanted patients with pure NK cells becomes a common clinical practice. NK cells isolated from donor blood and expanded effectively avoid infections and

Common sources for isolation of MSC are bone marrow aspirate, cord blood, and pieces of umbilical cord/placenta tissue/adipose tissue. MSC were originally identified in the 1970s from cellular suspensions from spleen and bone marrow by their capacity to adhere to plastic—which is still the standard form to culture MSCs and also by their ability to form colonies from single cells (explanted *ex vivo*), their fibroblast-like appearance and their capacity to differentiate into fat, cartilage, and bone. MSC are defined by surface markers of CD105, CD90, and CD73 expression, yet not CD45, CD34, and CD14 as of the consensus of ICSCT working group [46]. Recently, ICSCT also have defined a broad consensus of the international standards for harmonized potency assays to boost the clinical development of ATMP MSC therapy for many unmet clinical needs despite different tissue sources and disparate culture expansion protocols. Three preferred analytic methods in a matrix assay approach, namely, quantitative RNA analysis of selected gene products; flow cytometry analysis of functionally relevant surface markers, and protein-based assay of secretome have been proposed to reflect on the immunomodulatory potential of the ATMP cells for different clinical therapeutics as

well as to evolve the regulatory landscape for the sake of the progress in the field [47].

Several techniques are employed for liposuction used for adipose tissue-derived stromal cell collections [48]. Processed lipoaspirate (LPA) contains multipotent cells that can be an alternate stem cell source to bone-marrow-derived MSCs. LPA contains stromal vascular fraction (SVF) containing a number of different cell types such as adipose stromal cells (ASC), pericytes, endothelial cells, fibroblasts, preadipocytes, and hematopoietic stem cells. ASC have differentiation potential to myogenic, osteogenic, chondrogenic, or adipogenic on culturing with specific induction media [49]. SVF contains a lot of vascular cells and hematopoetic cells

For the isolation of MSC adherence of these cells, plastic surfaces are used. The usual procedure is to put the starting material into culture flasks or discs. After 10–20 days, nonadherent cells are washed out, and the adhered MSC colonies are passaged, suspended in a fresh medium, and seeded in new flasks for further expansion. Thus, expanded MSC have been used in all clinical trials (see **Table 7**). The unavoidable detaching procedures of MSC at passaging influence the receptor quality of MSC. Long-term cultivation of big numbers of MSC in bioreactors is possible and the provision of large seeding areas avoids unwanted differentiation of expanded MSC [54–57]. Procedures have been worked out to proceed directly into the expansion phase of MSC. Outgrowth and isolation of MSC can be successfully performed by giving BM aspirate into the sterile plastic vessels of perfusion bioreactors. MSC from BM aspirate are not only diverse tumor tissue preparations but can also be placed directly in ZRP meander perfusion bioreactors. MSC colonies or outgrown TIL from tumor tissue pieces can then further be

GvHD when applied immediately following transplantation.

252 Stem Cells in Clinical Practice and Tissue Engineering

that have to be eliminated before expansion of remaining MSC.

T cells are the most powerful immune cells. Despite the immense research on these cells enriching our deeper knowledge into T cell biology as well as their kinetics in health and diseases, the different T cell subsets are yet not routinely used as cell therapeutics. This is at least partly due to the complex nature of immune cells and many unsolved technical problems to produce and handle them. In **Tables 3**–**5**, some parameters have been compiled giving insight into methods and technologies as well as expansion success and purity of the different T cell types being used in clinical trials. The chosen examples in the tables are representative of the field and enlighten the diversity of production processes and produced cell specimen, and it explains probably in part the inconsistent and predominantly unsatisfying clinical results with more or less identical cell specimens.

Vaccination with dendritic cells being *ex vivo* treated with proteins or peptides from tumor cells or micro-organisms as well as present target-specific peptides to T cells *ex vivo*, directly


**Table 3.** Manufacturing processes of T cells in clinical trials (n.a.= not available).


derived) large number of generally stimulated T cells was of advantage for the treated patients could not be evidently established from published literatures. The example of CTL expansion and therapeutic use did not state details of the expansion process with missing information.

Landscape of Manufacturing Process of ATMP Cell Therapy Products for Unmet Clinical Needs

http://dx.doi.org/10.5772/intechopen.69335

To get effective, functional T cells out of PBMC expansion process have to be "conditional" (activation of sub-clones by specific cancer/micro-organism-derived structures; naive cell/cell and cell/matrix contact; preferred or suppressed growth of a cell population/sub population by specific cytokine/growth factor/antibody stimulation; controlled gas and nutrient supply). It is obvious from **Table 4** that the expansion of TIL even in bigger numbers is possible, up to more than 1010 TIL might be grown in flasks as well as in bags, G-rex flasks, Wave bioreactors, or ZRP perfusion bioreactors. Media and supplements are qualitatively similar. In all examples, use of feeder cells seem to be essential. Looking closer into **Table 4**, the fold expansion differs over a wide range. This implies that strongly differing numbers of TIL must have

been isolated. G-Rex flasks is of advantage for TIL expansion by providing high O<sup>2</sup>

value in the medium for TIL growth.

meander bioreactors, O2

optimal O2

**trials**

tion near to the sedimented TIL that enhances growth. In own studies (not published) of ZRP

Although a lot of clinical trials with TIL have been performed so far, there is not much information about clinical efficiency of TIL (with an exception of late-stage metastatic melanoma). Expanded TIL over a prolonged period often contains CD3+/CD4+/CD8+CD25+cells in different ratios. Deeper FACS analyses make likely that subclones that are contained are primed and directed against some single mutated clones. Again, it must be emphasized that even with TIL stronger standardized cell production processes, better characterization of contained clones, more comparable preclinical data as well as professional designed clinical trials are needed.

The expansion of several CAR-T cell specimen seems not to be a problem (**Table 5**). Blood-derived natural T cells were used for CAR transduction. Expansion to large numbers of the CAR-T cells was achieved by using the same interleukin activation and culturing procedures as in the case of normal T cells. Some of the advanced centers are engaged in optimizing newer technologies

including Miltenyi's CliniMACS Prodigy as well as Zellwerk's ZRP technology system.

**5. State-of-the-art with** *ex vivo* **expanded NK cells utilized in clinical** 

NK cells are getting more and more attention in fighting cancer and infections since it succeeded to expand these immune cells not only in huge amounts but also in pure quality [4–6, 61]. In contrast to T cells, NK cells do not show immunological incompatibility when administered in haploidentical or even allogenic clinical trials. It is, however, important that NK cells in such settings are totally free from T cells. In **Table 6**, a selection of clinical studies is listed, among them are those that are performed by known clinical research groups. It can be deduced that bigger numbers of NK cells can be produced in culture flaks as well as in some cell culture equipment consisting of plastic bags or vessels (VueLife static culturing in bags; wave system using different bags, mixing of medium by slow shaking movement of the bag). The bag systems are closed

(Zellwerk, Germany) concentration can be up-regulated to find an

concentra-

255

**Table 4.** Manufacturing processes of TIL in clinical trials (n.a.= not available).


**Table 5.** Manufacturing processes of CAR-T-cells in clinical trials (n.a.= not available).

or mediated by dendritic cells, and subsequent *ex vivo* expansion is used over a long time, and there are even a few cell therapeutics on the market. However, adjuvant treatment in different conditions of cancer in clinics show only limited success. In recent times, the isolation of TILs from tissue and/or microenvironment of solid tumors and expansion of TILs *ex vivo* has proven a much more promising way to get specific activated T cell in larger amounts.

From **Table 3**, it can be observed that the fold expansion in flasks and Wave bioreactor is similar, although the total number of T cells harvested differs enormously. Whether the (apheresis derived) large number of generally stimulated T cells was of advantage for the treated patients could not be evidently established from published literatures. The example of CTL expansion and therapeutic use did not state details of the expansion process with missing information.

To get effective, functional T cells out of PBMC expansion process have to be "conditional" (activation of sub-clones by specific cancer/micro-organism-derived structures; naive cell/cell and cell/matrix contact; preferred or suppressed growth of a cell population/sub population by specific cytokine/growth factor/antibody stimulation; controlled gas and nutrient supply).

It is obvious from **Table 4** that the expansion of TIL even in bigger numbers is possible, up to more than 1010 TIL might be grown in flasks as well as in bags, G-rex flasks, Wave bioreactors, or ZRP perfusion bioreactors. Media and supplements are qualitatively similar. In all examples, use of feeder cells seem to be essential. Looking closer into **Table 4**, the fold expansion differs over a wide range. This implies that strongly differing numbers of TIL must have been isolated. G-Rex flasks is of advantage for TIL expansion by providing high O<sup>2</sup> concentration near to the sedimented TIL that enhances growth. In own studies (not published) of ZRP meander bioreactors, O2 (Zellwerk, Germany) concentration can be up-regulated to find an optimal O2 value in the medium for TIL growth.

Although a lot of clinical trials with TIL have been performed so far, there is not much information about clinical efficiency of TIL (with an exception of late-stage metastatic melanoma). Expanded TIL over a prolonged period often contains CD3+/CD4+/CD8+CD25+cells in different ratios. Deeper FACS analyses make likely that subclones that are contained are primed and directed against some single mutated clones. Again, it must be emphasized that even with TIL stronger standardized cell production processes, better characterization of contained clones, more comparable preclinical data as well as professional designed clinical trials are needed.

The expansion of several CAR-T cell specimen seems not to be a problem (**Table 5**). Blood-derived natural T cells were used for CAR transduction. Expansion to large numbers of the CAR-T cells was achieved by using the same interleukin activation and culturing procedures as in the case of normal T cells. Some of the advanced centers are engaged in optimizing newer technologies including Miltenyi's CliniMACS Prodigy as well as Zellwerk's ZRP technology system.

### **5. State-of-the-art with** *ex vivo* **expanded NK cells utilized in clinical trials**

or mediated by dendritic cells, and subsequent *ex vivo* expansion is used over a long time, and there are even a few cell therapeutics on the market. However, adjuvant treatment in different conditions of cancer in clinics show only limited success. In recent times, the isolation of TILs from tissue and/or microenvironment of solid tumors and expansion of TILs *ex vivo* has

Bag ≤48 15.000 ~3×1011 CD4+: ≤82

From **Table 3**, it can be observed that the fold expansion in flasks and Wave bioreactor is similar, although the total number of T cells harvested differs enormously. Whether the (apheresis

proven a much more promising way to get specific activated T cell in larger amounts.

**Cell source; supplements; activation**

TIL, IL2, serum, feeder cells

TIL, IL2, serum, feeder cells, anti-CD3

TIL (6x GRex10) TIL, IL2,

TIL, IL2, feeder cells, anti CD3

TIL, IL2, feeder cells, anti CD3

TIL, serum, feeder cells, anti CD3, IL2, IL 15,

**Cell source; supplements, activation**

serum

IL-2

PBMC, anti-CD3, IL-2, feeder cells,

PBMC, anti-CD3, anti-CD28

PBMC, anti-CD3, IL-2, IL-15

PBMC, serum, feeder PBMCs,

IL21

**Expansion device used**

254 Stem Cells in Clinical Practice and Tissue Engineering

Flask Wave

GRex Flask

Bag Wave

Flask Wave

ZRP Meander bioreactor

**Expansion device used** **Cultivation time (days)**

24 72

29 180

>21 1259

>14 1433

**Table 4.** Manufacturing processes of TIL in clinical trials (n.a.= not available).

**Cultivation time (days)**

**Table 5.** Manufacturing processes of CAR-T-cells in clinical trials (n.a.= not available).

228

170

1130

5576

**Expansion by x-fold**

Well plates 14–24 n.a. n.a. (infusion:

**Expansion by x-fold**

Bag >14 1041 5 × 1010 n.a. [36]

0.4 × 1010 1.5 × 1010

4.5 × 1010 1.5 × 1010

≤1010)

Bag 12 10.6 n.a. CD3+: >95 [43]

WAVE 35 n.a. 109 n.a. [33]

>20 5000 2 × 109 CD8: ~60%

NA 62–72

**Cell harvest Cell purity (%) Reference**

35 + 52 CD4 + CD8 35 + 63

CD3+ CD8+ 62

CD3+: F = W CD4+: F > W CD8+: F < W

NA >97 [40]

CD4: 38% (High% Tem)

**Cell harvest Cell purity (%) Reference**

CD8+: ≤85

n.a. [41]

[37]

[38]

[39]

Own data, not published

[60]

NK cells are getting more and more attention in fighting cancer and infections since it succeeded to expand these immune cells not only in huge amounts but also in pure quality [4–6, 61]. In contrast to T cells, NK cells do not show immunological incompatibility when administered in haploidentical or even allogenic clinical trials. It is, however, important that NK cells in such settings are totally free from T cells. In **Table 6**, a selection of clinical studies is listed, among them are those that are performed by known clinical research groups. It can be deduced that bigger numbers of NK cells can be produced in culture flaks as well as in some cell culture equipment consisting of plastic bags or vessels (VueLife static culturing in bags; wave system using different bags, mixing of medium by slow shaking movement of the bag). The bag systems are closed systems, and some of the equipment are provided with regulating and/or steering elements for gasing, measurement of pH, pO<sup>2</sup> , and medium temperature is obligatory. However, changing bag volumes and upscaling within a cultivation run is not easy and possible with these systems. One main advantage of NK cells is the good compatibility of this immune cell specimen. NK cells can be expanded up to 50,000-fold in suited perfusion bioreactors (**Table 6**), whereas this is by far not achieved in culture flasks. Adjuvant treatment of stem cell-transplanted patients with pure NK cells becomes common. NK cells isolated from donor blood and expanded avoids effectively GvHD when applied during the first phase after transplantation.

production of MSC for adequate amounts of immune cells needs specific attention. Most of the current clinical trials use open culture system in flasks even though there have been ongo-

Landscape of Manufacturing Process of ATMP Cell Therapy Products for Unmet Clinical Needs

Consistent and logistically practical methods for production of MSC for adequate numbers of immune cells need specific attention. In addition, there are many unresolved issues relating to the isolation, expansion technique, phenotyping characterization, mechanisms of action, and incomparability of study results due to different protocols and definitions. In most of the current clinical trials, open culture system in flasks is in use (compare **Table 7**) even though there have been ongoing efforts with newer technologies [56, 70, 71]. In contrast, MSC, in particular those derived from bone marrow, lead candidates to fight many important disease entities. It is believed that modulation of immune responses, remodeling of impaired tissues, pro-regenerative as well as

were reported in treatment of some autoimmune and infectious diseases [68, 69]. A lot of clinical trials have been performed or are ongoing on treating cardiovascular diseases (myocardial infarction, cardiomyopathy, critical limb ischemia, stroke [72–74]). Single or repeated doses of

cardiovascular disease could be ensured through cytokines and chemokines secreted from MSC. Avoiding GvHD is also under intense investigation due to increasing stem cell transplantation in cancer and organ transplant patients. In all these indications, autologous or allogenic MSC are

> **Expansion by x-fold**

28 5–145 2 x 107

**7. Newer techniques for characterization and production of immune** 

Identification of different cell types, subpopulations, and even single subclones within a final cell therapeutics product can be a challenging exercise due to many constraints including the limited number of cells available. Immune cells are usually identified from its displayed surface receptors that also gives a hint on its characters to fight infections or cancer. Cell sorting by flow cytometry or magnetic beads are modern techniques allowing isolation and separation

are injected systemically or topically. Until now, some positive influences on

10–28 n.a. 6–27 x 107 n.a. [52]

x 109

22–28 n.a. 1–8 x 108 n.a. [52]

to 5

**Cell harvest Cell purity Reference**

http://dx.doi.org/10.5772/intechopen.69335

n.a. [53]

MSC, effects

257

antifibrotic effects can be attained with these immune cells. With numbers of 10<sup>9</sup>

being infused that are not really manufactured under controlled conditions.

**Cultivation time (days)**

BM-MNC, serum Flasks 30 n.a. n.a. 99% [50] BM-MNC, serum Flasks 30–45 6–52 2.4–5.7 x 107 >60% [51]

**Expansion device used**

flasks

flasks

Five layer flasks

**Table 7.** Manufacturing processes of MSC in clinical trials (n.a.= not available).

ing efforts with newer technologies (compare **Table 7**).

107

**Cell source; supplements**

BM-W, serum 59 donors

to more than 109

BM-E serum Five layer

UC blood, serum FIve layer

**cells therapeutics**

Advanced production technology makes NK cells attractive for use in broader bases: Pure NK cells have shown nearly no unwanted side effects in clinical trials even when administered in high doses. The modern production processes deliver NK cells with enhanced functionalities (high cytotoxicity against many cancer cells in *ex vivo* tests and enhanced paracrine production). Pure NK cells can be manufactured in an easy-to-handle closed system as ATMP in clinical settings near to patients. A particular advantage is that mass amounts of individual NK cells can be produced economically (due to low costs of selection, medium, and activation).


**Table 6.** NK cells for cell therapies. Different production methods.

### **6. State-of-the-art with** *ex vivo* **expanded MSCs utilized in clinical trials**

With numbers of 109 MSC, therapeutic effects were reported in the treatment of autoimmune and infectious diseases [68, 69]. The issue on reliabilities and logistically practical methods for production of MSC for adequate amounts of immune cells needs specific attention. Most of the current clinical trials use open culture system in flasks even though there have been ongoing efforts with newer technologies (compare **Table 7**).

systems, and some of the equipment are provided with regulating and/or steering elements for

bag volumes and upscaling within a cultivation run is not easy and possible with these systems. One main advantage of NK cells is the good compatibility of this immune cell specimen. NK cells can be expanded up to 50,000-fold in suited perfusion bioreactors (**Table 6**), whereas this is by far not achieved in culture flasks. Adjuvant treatment of stem cell-transplanted patients with pure NK cells becomes common. NK cells isolated from donor blood and expanded avoids

Advanced production technology makes NK cells attractive for use in broader bases: Pure NK cells have shown nearly no unwanted side effects in clinical trials even when administered in high doses. The modern production processes deliver NK cells with enhanced functionalities (high cytotoxicity against many cancer cells in *ex vivo* tests and enhanced paracrine production). Pure NK cells can be manufactured in an easy-to-handle closed system as ATMP in clinical settings near to patients. A particular advantage is that mass amounts of individual NK cells can be produced economically (due to low costs of selection, medium, and activation).

> **Cultivation time (days)**

**6. State-of-the-art with** *ex vivo* **expanded MSCs utilized in clinical trials**

and infectious diseases [68, 69]. The issue on reliabilities and logistically practical methods for

MSC, therapeutic effects were reported in the treatment of autoimmune

effectively GvHD when applied during the first phase after transplantation.

**Expansion device** 

**used**

(750 ml)

VueLife bag system (800 ml)

VueLife bag system

ZRP type M singleuse bioreactor

ZRP typ M singleuse bioreactor

**Table 6.** NK cells for cell therapies. Different production methods.

PBMC fraction, serum; IL 2, Stirred bioreactor

, and medium temperature is obligatory. However, changing

**Expansion by x-fold**

33 352 96% [62]

14 80–200 85–91% [63]

21 277 97% [65]

30 1000–2000 95–99% Own data, not

30–35 1000–50,000 99% Own data, not

T-flaks 10 40 62–95% [64]

Wave bioreactor 21 12–354 37–54% [66]

G-Rex flask 8–10 442 54–79 [67]

**Cell purity Reference**

published

published

gasing, measurement of pH, pO<sup>2</sup>

256 Stem Cells in Clinical Practice and Tissue Engineering

**Cell source; supplements,** 

PBMC fraction; serum; IL 2;IL

PBMC fraction; serum; IL2/ IL12/IL15; feeder cell line

PBMC fraction; serum; IL 2; IL 15; feeder cell line K529; 4

PBMC fraction; medium;

PBMC fraction; medium; serum; IL 2; feeder cell line

PBMC fraction; medium;

PBMC fraction; serum; IL 2/IL 21; coating with CD 16

**activation**

15; PHA

BBL1

K529;

serum; IL 2

serum; IL 2;

With numbers of 109

Consistent and logistically practical methods for production of MSC for adequate numbers of immune cells need specific attention. In addition, there are many unresolved issues relating to the isolation, expansion technique, phenotyping characterization, mechanisms of action, and incomparability of study results due to different protocols and definitions. In most of the current clinical trials, open culture system in flasks is in use (compare **Table 7**) even though there have been ongoing efforts with newer technologies [56, 70, 71]. In contrast, MSC, in particular those derived from bone marrow, lead candidates to fight many important disease entities. It is believed that modulation of immune responses, remodeling of impaired tissues, pro-regenerative as well as antifibrotic effects can be attained with these immune cells. With numbers of 10<sup>9</sup> MSC, effects were reported in treatment of some autoimmune and infectious diseases [68, 69]. A lot of clinical trials have been performed or are ongoing on treating cardiovascular diseases (myocardial infarction, cardiomyopathy, critical limb ischemia, stroke [72–74]). Single or repeated doses of 107 to more than 109 are injected systemically or topically. Until now, some positive influences on cardiovascular disease could be ensured through cytokines and chemokines secreted from MSC. Avoiding GvHD is also under intense investigation due to increasing stem cell transplantation in cancer and organ transplant patients. In all these indications, autologous or allogenic MSC are being infused that are not really manufactured under controlled conditions.


**Table 7.** Manufacturing processes of MSC in clinical trials (n.a.= not available).

### **7. Newer techniques for characterization and production of immune cells therapeutics**

Identification of different cell types, subpopulations, and even single subclones within a final cell therapeutics product can be a challenging exercise due to many constraints including the limited number of cells available. Immune cells are usually identified from its displayed surface receptors that also gives a hint on its characters to fight infections or cancer. Cell sorting by flow cytometry or magnetic beads are modern techniques allowing isolation and separation of immune cell subtypes. These methods are still time-consuming and costly exercises due to the quantity of antibodies and reagents required in the manufacturing process when dealing with larger starting material such as cells derived through apheresis. GMP-grade antibodies are particularly costly making the ATMP cell therapy an unaffordable range.

CD4+ as well as CD8+ T cells, NK cells, DC cells, and activated B cells seems to be essential for achieving sustainable effects to eradicate tumors with immune cell therapy. This has much

Landscape of Manufacturing Process of ATMP Cell Therapy Products for Unmet Clinical Needs

http://dx.doi.org/10.5772/intechopen.69335

The ZRP platform of Zellwerk and the belonging bioreactor types provide sophisticated features not only for mass production of different immune cells but also for realizing isolation and culturing the cells in closed processes [54–57, 70, 75]. It enables controlled phases of selection/priming/activation of seeded cells by regulated medium flow, suited coating of seeding surfaces and/or fixed antibodies, followed by rapid expansion, all in a single run. This is due

Bioreactors of the ZRP system can be operated in the GMP breeder (**Figure 1**). The breeder combines a laminar flow sterile bench and an incubator. Controlling of the essential bioreac-

The cell cultivation platform and the belonging bioreactors enable the manufacturing of large quantities of individual immune cell preparations under GMP conditions. A series of ZRP systems can be driven in parallel in one clean room equipment due to the closed steps of the perfusion bioreactor processes. During a period of 1 year, several immune cell preparations from individuals amounting to ~100 can be undertaken in one B clean room thus reducing expenditures for production of immune cell therapeutics massive. Manufacturing of immune

Important features of the meander type bioreactors are as follows: A directed laminar flow of medium, which can be chosen over a wide range, makes an undisturbed cell/cell- and cell/surface-contact possible and minimizes cell stress. The ratio of medium circulation and fresh medium flow is automatically regulated over time by a chosen algorithm guaranteeing ananan consistent homogenous supply with nutrients and gasses as well as precise regulated

, pH, and temperature in the medium. T cells, TILs, and NK cells can be expanded to more than a 1010 cells in one closed cultivation run. In parallel cultivation run with pure NK

, pH, medium

259

tor and breeder functions is by the control unit (automatic regulation of pO2

temperature, medium feeding, mixing, and flow of gasses over a touch screen).

cells as ATMP is authorized by the national and regional German authorities.

**Figure 1.** Zellwerk's GMP Z®RP cell breeder with M type bioreactor for NK/T/TIL cells.

more connotation for the ATMP cell therapy to reach its full potential.

to the technical attributes:

pO2

However, it is pertinent to analyze and to predict the potency of the cells that give a prior indication of the anticipated possible effects of the immune cells on the intended outcome. In case of T cells, it has been recently accomplished by tracking the fate and origin of clinically relevant adoptively transferred CD8+ T cells *in vivo* to identify and track single subclones specifically activated against few tumor cell mutants specific T memory cells [30]. By using highthroughput T cell receptor sequencing, the group has worked out a strategy to identify and track those very low frequency monoclonal T cells among the total bulk of polyclonal T cell pool with varying cancer-killing and fighting capabilities that have been given as adoptive cell therapy to 10 metastatic malignant melanoma patients being specifically activated against melanoma and correlated with the treatment response in patients. They were then also able to decipher the specific clonal population of extremely low density of T cells that were persisting and effective *in vivo* among two patients out of ten demonstrating complete remission. It is worth proving whether this new approach can be applied effectively in clinical practice and to prove its rationale in other cancer types. Only around 0.001% of all T cells in blood consisted of these active monoclonal T cells. This study also indicated that the younger T cells nearly in the phase of development had a better ability to fight tumors than older ones.

Further progress in the identification of subpopulations, primed monoclonal T cells, and information on functionality of cell preparations may be obtained by the spectra of single cells, their typical receptors/ligands/paracrine production. Raman microscopy has long been used in cell and metabolites analysis. However, the combination of this method with sophisticated software programs with in-depth analyses tools can lead to sharper, high-resolution Raman spectra enabling differentiating looks onto cells enabling subtype identification, quantification, analysis of functional status, etc. [10–13].

CyTOF, the latest novel format of flow cytometry combined with mass spectrometry, often referred as mass cytometry provides a measurement of >40 simultaneous cellular parameters at single-cell resolution, significantly augmenting the ability of cytometry to evaluate complex cellular systems and processes at any given point. This has been a greatest tool to unravel the mechanism of immune cells by studying kinetics before and after infusion. Currently, research on solid cancers has a strong focus on immune cells infiltrations in the microenvironment of tumors. In a mouse model of triple negative breast carcinoma, the new methods using CyTOF assessing immune cells at single cell level within the tumor in the microenvironment of the tumor as well systemically in different organs over time demonstrated a striking difference of the sustained response at systemic level in the effective treatment responsive group in comparison to the nonresponsive treatment group [31]. Local carcinoma treatment was followed by an infiltration of CD8+/CD4+ T cells into the tumor leading to death of tumor cells. Different immune cell specimens were expanded during the rejection phase not only in the microenvironment of the carcinoma but also in many lymphatic organs and blood to reflect on the changes taking place systemically. A systemic coordinated immune response of CD4+ as well as CD8+ T cells, NK cells, DC cells, and activated B cells seems to be essential for achieving sustainable effects to eradicate tumors with immune cell therapy. This has much more connotation for the ATMP cell therapy to reach its full potential.

of immune cell subtypes. These methods are still time-consuming and costly exercises due to the quantity of antibodies and reagents required in the manufacturing process when dealing with larger starting material such as cells derived through apheresis. GMP-grade antibodies

However, it is pertinent to analyze and to predict the potency of the cells that give a prior indication of the anticipated possible effects of the immune cells on the intended outcome. In case of T cells, it has been recently accomplished by tracking the fate and origin of clinically relevant adoptively transferred CD8+ T cells *in vivo* to identify and track single subclones specifically activated against few tumor cell mutants specific T memory cells [30]. By using highthroughput T cell receptor sequencing, the group has worked out a strategy to identify and track those very low frequency monoclonal T cells among the total bulk of polyclonal T cell pool with varying cancer-killing and fighting capabilities that have been given as adoptive cell therapy to 10 metastatic malignant melanoma patients being specifically activated against melanoma and correlated with the treatment response in patients. They were then also able to decipher the specific clonal population of extremely low density of T cells that were persisting and effective *in vivo* among two patients out of ten demonstrating complete remission. It is worth proving whether this new approach can be applied effectively in clinical practice and to prove its rationale in other cancer types. Only around 0.001% of all T cells in blood consisted of these active monoclonal T cells. This study also indicated that the younger T cells nearly in

are particularly costly making the ATMP cell therapy an unaffordable range.

258 Stem Cells in Clinical Practice and Tissue Engineering

the phase of development had a better ability to fight tumors than older ones.

tification, analysis of functional status, etc. [10–13].

Further progress in the identification of subpopulations, primed monoclonal T cells, and information on functionality of cell preparations may be obtained by the spectra of single cells, their typical receptors/ligands/paracrine production. Raman microscopy has long been used in cell and metabolites analysis. However, the combination of this method with sophisticated software programs with in-depth analyses tools can lead to sharper, high-resolution Raman spectra enabling differentiating looks onto cells enabling subtype identification, quan-

CyTOF, the latest novel format of flow cytometry combined with mass spectrometry, often referred as mass cytometry provides a measurement of >40 simultaneous cellular parameters at single-cell resolution, significantly augmenting the ability of cytometry to evaluate complex cellular systems and processes at any given point. This has been a greatest tool to unravel the mechanism of immune cells by studying kinetics before and after infusion. Currently, research on solid cancers has a strong focus on immune cells infiltrations in the microenvironment of tumors. In a mouse model of triple negative breast carcinoma, the new methods using CyTOF assessing immune cells at single cell level within the tumor in the microenvironment of the tumor as well systemically in different organs over time demonstrated a striking difference of the sustained response at systemic level in the effective treatment responsive group in comparison to the nonresponsive treatment group [31]. Local carcinoma treatment was followed by an infiltration of CD8+/CD4+ T cells into the tumor leading to death of tumor cells. Different immune cell specimens were expanded during the rejection phase not only in the microenvironment of the carcinoma but also in many lymphatic organs and blood to reflect on the changes taking place systemically. A systemic coordinated immune response of The ZRP platform of Zellwerk and the belonging bioreactor types provide sophisticated features not only for mass production of different immune cells but also for realizing isolation and culturing the cells in closed processes [54–57, 70, 75]. It enables controlled phases of selection/priming/activation of seeded cells by regulated medium flow, suited coating of seeding surfaces and/or fixed antibodies, followed by rapid expansion, all in a single run. This is due to the technical attributes:

Bioreactors of the ZRP system can be operated in the GMP breeder (**Figure 1**). The breeder combines a laminar flow sterile bench and an incubator. Controlling of the essential bioreactor and breeder functions is by the control unit (automatic regulation of pO2 , pH, medium temperature, medium feeding, mixing, and flow of gasses over a touch screen).

The cell cultivation platform and the belonging bioreactors enable the manufacturing of large quantities of individual immune cell preparations under GMP conditions. A series of ZRP systems can be driven in parallel in one clean room equipment due to the closed steps of the perfusion bioreactor processes. During a period of 1 year, several immune cell preparations from individuals amounting to ~100 can be undertaken in one B clean room thus reducing expenditures for production of immune cell therapeutics massive. Manufacturing of immune cells as ATMP is authorized by the national and regional German authorities.

Important features of the meander type bioreactors are as follows: A directed laminar flow of medium, which can be chosen over a wide range, makes an undisturbed cell/cell- and cell/surface-contact possible and minimizes cell stress. The ratio of medium circulation and fresh medium flow is automatically regulated over time by a chosen algorithm guaranteeing ananan consistent homogenous supply with nutrients and gasses as well as precise regulated pO2 , pH, and temperature in the medium. T cells, TILs, and NK cells can be expanded to more than a 1010 cells in one closed cultivation run. In parallel cultivation run with pure NK

**Figure 1.** Zellwerk's GMP Z®RP cell breeder with M type bioreactor for NK/T/TIL cells.

cells using the same medium and density of seeded cells, expansion in static flasks was not more than 50–100-fold, whereas in meander type bioreactor 5000–50,000-fold was achieved. Coating of seeding areas with specific matrices/antibodies, i.e., can be exploited to promote suppression or expansion of several immune cell specimen (e.g., Treg; CD56bright NK cells).

closed automated hollow fiber bioreactor system for GMP cell manufacturing that has been tried in MSC expansion [82]. Huge efforts are ongoing in Japan to build modular roboticsmanipulated, automated high-throughput cell-processing machine to make off-the-shelf cell

Landscape of Manufacturing Process of ATMP Cell Therapy Products for Unmet Clinical Needs

http://dx.doi.org/10.5772/intechopen.69335

261

Product dictates the process development and optimization to ensure cost-effectiveness, wellcontrolled steps, high reliability, high cell density and viability, high product quality, easy recovery, high yield, and high safety for personnel. Perfusion system takes care of controlled dynamic gas exchange with a homogenous environment allowing high cell density with controllable and flexible process control, but demands complicated validation procedure.

Evolving regulatory landscape for ATMPs (cell therapy) [84–87] has the following two man-

• Cells or tissues that have been subjected to substantial manipulation so that biological characteristics, physiological functions, or structural properties relevant for the intended clinical use have been altered, or of cells or tissues that are not intended to be used for the same

• Have properties for, or are used in or administered to human beings with a view to treating, preventing, or diagnosing a disease through the pharmacological, immunological, or

Regulatory landscape for ATMP cell therapy at this juncture is quite diverse in various regions of the world and is evolving though in a gradient [89–95]. It is a daunting task, but is an absolute necessity to harmonize all the harmonizers (regulatory bodies) across the globe with an unified coherent process to make ATMP cell therapy possible for unmet clinical needs. The regulatory bodies have the responsibilities to assure the safety and rights of patients and to ensure quality of the nonclinical and clinical evidences to allow appropriate evaluation of the safety and effectiveness of the cell therapy product through clinical trials for market authorization. FDA and EMA have distinct guidelines, whereas there are distinct guidelines in other parts such as Australia, Brazil, Japan, Korea, Singapore, and Taiwan just to give a flavor of the unique differences that pose constraints in the current clinical development of ATMP cell therapy. While such efforts to converge on an unified regulatory process are underway through engagements, two critical issues have been identified to be addressed, the concept of

FDA has developed a number of expedited programs to facilitate ATMP cell therapy use in patients when no satisfactory alternative therapies are available while ensuring the standards of the products for safety and efficacy. EMA made a new regulation in 2009 for all 28 member countries in EU with obligatory centralized market authorization process. Eight cell therapy ATMPs have been authorized in EU [97], namely ChondroCelect (withdrawn in January 2017) and Maci (suspended from July 2014 due to closure of European manufacturing unit), both the products are autologous cartilage cells grown *ex vivo* for cartilage repair; Provenge (autologous PBMC activated with fusion protein of prostatic acid phosphatase attached to GM-CSF *ex vivo*) for treatment of advanced prostate cancer, withdrawn in May

therapy products for wider application [83].

dates to be fulfilled (quoted from EMA document [88]):

essential function(s) in the recipient and the donor.

potency assessment as well as *in vivo* tumorigenicity studies [96].

metabolic action of their cells or tissues.

### **8. Current advances and future prospects**

Despite many clinical trials with immune cell therapy and much progresses in the field, the manufacturing of cells as ATMP for routine clinical use has not been realized in its full potential. Here we attempt to give an overview of different advanced technologies and devices currently available for cell expansion. These different devices need to be validated and compared in head-to-head comparison for different cell type to exploit the opportunities for unmet clinical needs.

Sterility, purity, identity, and potency are four cardinal requirements. Initially, cell expansions have been carried out in the conventional process of open flask culture system with gradual incremental innovations to meet the growing needs of regulatory requirements as well as better quality products in terms of safety (phenotype), consistency (reproducibility and controlled robust process), numbers, quality (structural and functional), and efficacy (functionality and potency) to qualify as ATMP to be used in patients/human subjects. From simple culture flasks, multilayered flask was made for ease of handling as well as scaling up with the numbers (such as Millicell, Millipore or BD Multiflask).

Different microcarrier system came into place to increase surface area to expand cells in 3D than in flasks in 2D [76–78]. G-Rex system is a gas permeable rapid expansion device with a silicone membrane at the base, allowing gas exchange to occur uninhibited by the depth of the medium above with high O2 concentration stimulating cell expansion and simplify handling [99]. WAVE-type bioreactor systems [79] make use of sterile, transparent, and disposable culture bags with provision for media perfusion, harvest, sampling, and gas exchange, which greatly reduces the cross-contamination problem, one of the biggest challenge in conventional culture system [37, 38]. The culture bag is put on the device's temperature-controlled tray inside an incubator having the option of controlled rocking movement. Optional perfusion modules enable controlled addition and removal of culture media for optimized nutrient concentrations while disposing spent media resulting in higher cell densities with involvement of less time and effort for media exchange. Magnetic beads are used in the bag for various priming and activation of cells [80]. However, the chaotic medium flow causes cell stress [78, 79]. These factors have been well addressed in ZRP cell breeder that has controlled and directed medium flow and fulfills most of the regulatory requirements (see Section 7).

MILTENYI's CliniMACS Prodigy® [81] is an automated integrated sensor-controlled closed system device that uses single-use sterile disposable tubing set and can perform fractionation of cells, cell washing, cell separation, cell culture, and final product formulation in the workflow connected through sterile docking devices. TERUMO quantum system is a functionally closed automated hollow fiber bioreactor system for GMP cell manufacturing that has been tried in MSC expansion [82]. Huge efforts are ongoing in Japan to build modular roboticsmanipulated, automated high-throughput cell-processing machine to make off-the-shelf cell therapy products for wider application [83].

cells using the same medium and density of seeded cells, expansion in static flasks was not more than 50–100-fold, whereas in meander type bioreactor 5000–50,000-fold was achieved. Coating of seeding areas with specific matrices/antibodies, i.e., can be exploited to promote suppression or expansion of several immune cell specimen (e.g., Treg; CD56bright NK cells).

Despite many clinical trials with immune cell therapy and much progresses in the field, the manufacturing of cells as ATMP for routine clinical use has not been realized in its full potential. Here we attempt to give an overview of different advanced technologies and devices currently available for cell expansion. These different devices need to be validated and compared in head-to-head comparison for different cell type to exploit the opportunities for unmet clini-

Sterility, purity, identity, and potency are four cardinal requirements. Initially, cell expansions have been carried out in the conventional process of open flask culture system with gradual incremental innovations to meet the growing needs of regulatory requirements as well as better quality products in terms of safety (phenotype), consistency (reproducibility and controlled robust process), numbers, quality (structural and functional), and efficacy (functionality and potency) to qualify as ATMP to be used in patients/human subjects. From simple culture flasks, multilayered flask was made for ease of handling as well as scaling up

Different microcarrier system came into place to increase surface area to expand cells in 3D than in flasks in 2D [76–78]. G-Rex system is a gas permeable rapid expansion device with a silicone membrane at the base, allowing gas exchange to occur uninhibited by the depth of the

[99]. WAVE-type bioreactor systems [79] make use of sterile, transparent, and disposable culture bags with provision for media perfusion, harvest, sampling, and gas exchange, which greatly reduces the cross-contamination problem, one of the biggest challenge in conventional culture system [37, 38]. The culture bag is put on the device's temperature-controlled tray inside an incubator having the option of controlled rocking movement. Optional perfusion modules enable controlled addition and removal of culture media for optimized nutrient concentrations while disposing spent media resulting in higher cell densities with involvement of less time and effort for media exchange. Magnetic beads are used in the bag for various priming and activation of cells [80]. However, the chaotic medium flow causes cell stress [78, 79]. These factors have been well addressed in ZRP cell breeder that has controlled and directed

MILTENYI's CliniMACS Prodigy® [81] is an automated integrated sensor-controlled closed system device that uses single-use sterile disposable tubing set and can perform fractionation of cells, cell washing, cell separation, cell culture, and final product formulation in the workflow connected through sterile docking devices. TERUMO quantum system is a functionally

medium flow and fulfills most of the regulatory requirements (see Section 7).

concentration stimulating cell expansion and simplify handling

**8. Current advances and future prospects**

260 Stem Cells in Clinical Practice and Tissue Engineering

with the numbers (such as Millicell, Millipore or BD Multiflask).

cal needs.

medium above with high O2

Product dictates the process development and optimization to ensure cost-effectiveness, wellcontrolled steps, high reliability, high cell density and viability, high product quality, easy recovery, high yield, and high safety for personnel. Perfusion system takes care of controlled dynamic gas exchange with a homogenous environment allowing high cell density with controllable and flexible process control, but demands complicated validation procedure.

Evolving regulatory landscape for ATMPs (cell therapy) [84–87] has the following two mandates to be fulfilled (quoted from EMA document [88]):


Regulatory landscape for ATMP cell therapy at this juncture is quite diverse in various regions of the world and is evolving though in a gradient [89–95]. It is a daunting task, but is an absolute necessity to harmonize all the harmonizers (regulatory bodies) across the globe with an unified coherent process to make ATMP cell therapy possible for unmet clinical needs. The regulatory bodies have the responsibilities to assure the safety and rights of patients and to ensure quality of the nonclinical and clinical evidences to allow appropriate evaluation of the safety and effectiveness of the cell therapy product through clinical trials for market authorization. FDA and EMA have distinct guidelines, whereas there are distinct guidelines in other parts such as Australia, Brazil, Japan, Korea, Singapore, and Taiwan just to give a flavor of the unique differences that pose constraints in the current clinical development of ATMP cell therapy. While such efforts to converge on an unified regulatory process are underway through engagements, two critical issues have been identified to be addressed, the concept of potency assessment as well as *in vivo* tumorigenicity studies [96].

FDA has developed a number of expedited programs to facilitate ATMP cell therapy use in patients when no satisfactory alternative therapies are available while ensuring the standards of the products for safety and efficacy. EMA made a new regulation in 2009 for all 28 member countries in EU with obligatory centralized market authorization process. Eight cell therapy ATMPs have been authorized in EU [97], namely ChondroCelect (withdrawn in January 2017) and Maci (suspended from July 2014 due to closure of European manufacturing unit), both the products are autologous cartilage cells grown *ex vivo* for cartilage repair; Provenge (autologous PBMC activated with fusion protein of prostatic acid phosphatase attached to GM-CSF *ex vivo*) for treatment of advanced prostate cancer, withdrawn in May 2015; Holoclar, autologous limbal stem cells to repair damaged corneal epithelium, this has orphan drug status due to rare condition. A great deal of effort are underway in EMA to refine and execute adaptive regulatory pathway to foster rapid development and accelerated assessment for innovative cell therapies. Korea has 14 cell products authorized including four stem cell products with 46 ongoing clinical trials with other cell therapy products. New adaptive regulatory framework has been enacted under the Pharmaceuticals, Medical Devices and Other Therapeutic Products Act (PMD Act) in Japan in late 2014 to facilitate access of promising ATMP cell therapy to the patients with limited treatment options as well as creating conducive regulatory environment to give accelerated conditional and time-limited authorization to stimulate further clinical development. Two products namely, MSC for GvHD second-line therapy and skeletal myoblast sheets for ischaemic heart failure have been authorized under the new scheme in 2015 and 2016.

**Conflict of Interest**

**Author details**

Hamburg, Germany

\*, Shreemanta K. Parida2

\*Address all correspondence to: poertner@tuhh.de

3Zellwerk GmbH, Oberkrämer, Germany

[1] ClinicalTrials Registry. USA: NIH; 2017

31036. DOI: 10.3402/jmahp.v4.31036

6066.CIR-16-0197

2016;**130**:279-294

DOI: 10.1155/2011/676198

Education Book. 2013;**1**:247-253

Hematology & Oncology. 2017;**10**:53

Ralf Pörtner1

**References**

GmbH.

All authors RP, SKP and CS do not have any conflict of interest. HH is the CEO of Zellwerk

Landscape of Manufacturing Process of ATMP Cell Therapy Products for Unmet Clinical Needs

, Christiane Schaffer<sup>1</sup>

[2] Hanna E, Remuzat C, Auquier P, Toumi M. Advanced therapy medicinal products: Current and future perspectives. Journal of Market Access & Health Policy. 2016;**4**:

[3] Galluzzi L, Zitvogel L, Kroemer G. Immunological mechanisms underneath the efficacy of cancer therapy. Cancer Immunology Research. 2016;**4**:895-902. DOI: 10.1158/2326-

[4] Koepsell SA, Miller JS, McKenna DH. Natural killer cells: A review of manufacturing

[5] Levy EM, Roberti MP, Mordoh J. Natural killer cells in human cancer: From biological functions to clinical applications. Journal of Biomedicine & Biotechnology. 2011;**676198**.

[6] Miller JS. Therapeutic applications: Natural killer cells in the clinic. Hematology. ASH

[7] Yang JC, Rosenberg SA. Adoptive T-cell therapy for cancer. Advances in Immunology.

[8] Wang Z, Wu Z, Liu Y, Han W. New development in CAR-T cell therapy. Journal of

and clinical utility. Transfusion. 2013;**53**:404-410. DOI: 10.1155/2011/676198

1 Hamburg University of Technology, Institute of Bioprocess and Biosystems Engineering,

2 Independent Global Health and Translational Medicine Consultant, Berlin, Germany

and Hans Hoffmeister<sup>3</sup>

http://dx.doi.org/10.5772/intechopen.69335

263

Since cell culture uses animal-derived serum or growth factors, the sources need to be certified and proven free of any adventitious agents, consistent in quality and free of risk of any possible infections.

First ATMP cell therapy product to get market approval was in Canada for Prochymal (Remestemcel-L), adult MSC for IV infusion for acute GvHD in May 2012. Allogeneic adipose tissue-derived MSC expanded *ex vivo* have been shown efficacious in Phase 3 clinical trial when given intra-lesionally in complex perianal fistulas in Crohn's disease patients and a decision is awaited in 2017 for market authorization by EMA. This product named as Cx601 by Takeda and TiGenix has received orphan status by the Swiss Agency for Therapeutic Products (Swissmedic) for the rare disease. While these innovations are taking place at immense pace, there is growing requirement of policymakers to be engaged along with patients' community to see how best a value-based frameworks and be drawn as rational approaches to use these expensive novel therapeutic modalities in patient care [98].

### **9. Conclusion**

The manufacturing of immune cells is until today primarily performed using archaic, scarcely controlled, incomparable processes and methods. These issues need to be better harmonized and put into standard practice. When looking into the processes of immune cell production used in clinical studies, it is obvious that the cells in most settings are expanded totally or partly in conventional culture flasks or similar vessels. That is due to the fact, that all immune cell types can be grown in this simple and cheap way without special skills. However, deeper characterization of *ex vivo* expanded immune cells is urgently needed not only on the level of a few receptors and ligands on the cell surface but also with respect to the ever-contained subtypes in an expanded immune cell population, the pattern of secreted effector molecules, their amounts over time and influences from *in vivo* components on them.

More research on aspects of modern cell therapy might be qualified as too costly, but will be more targeted and will at least avoid expensive and unjustified clinical studies maximizing the best use of the available R&D resources for better outcomes.

### **Conflict of Interest**

2015; Holoclar, autologous limbal stem cells to repair damaged corneal epithelium, this has orphan drug status due to rare condition. A great deal of effort are underway in EMA to refine and execute adaptive regulatory pathway to foster rapid development and accelerated assessment for innovative cell therapies. Korea has 14 cell products authorized including four stem cell products with 46 ongoing clinical trials with other cell therapy products. New adaptive regulatory framework has been enacted under the Pharmaceuticals, Medical Devices and Other Therapeutic Products Act (PMD Act) in Japan in late 2014 to facilitate access of promising ATMP cell therapy to the patients with limited treatment options as well as creating conducive regulatory environment to give accelerated conditional and time-limited authorization to stimulate further clinical development. Two products namely, MSC for GvHD second-line therapy and skeletal myoblast sheets for ischaemic heart failure have been

Since cell culture uses animal-derived serum or growth factors, the sources need to be certified and proven free of any adventitious agents, consistent in quality and free of risk of any

First ATMP cell therapy product to get market approval was in Canada for Prochymal (Remestemcel-L), adult MSC for IV infusion for acute GvHD in May 2012. Allogeneic adipose tissue-derived MSC expanded *ex vivo* have been shown efficacious in Phase 3 clinical trial when given intra-lesionally in complex perianal fistulas in Crohn's disease patients and a decision is awaited in 2017 for market authorization by EMA. This product named as Cx601 by Takeda and TiGenix has received orphan status by the Swiss Agency for Therapeutic Products (Swissmedic) for the rare disease. While these innovations are taking place at immense pace, there is growing requirement of policymakers to be engaged along with patients' community to see how best a value-based frameworks and be drawn as rational approaches to use these

The manufacturing of immune cells is until today primarily performed using archaic, scarcely controlled, incomparable processes and methods. These issues need to be better harmonized and put into standard practice. When looking into the processes of immune cell production used in clinical studies, it is obvious that the cells in most settings are expanded totally or partly in conventional culture flasks or similar vessels. That is due to the fact, that all immune cell types can be grown in this simple and cheap way without special skills. However, deeper characterization of *ex vivo* expanded immune cells is urgently needed not only on the level of a few receptors and ligands on the cell surface but also with respect to the ever-contained subtypes in an expanded immune cell population, the pattern of secreted effector molecules,

More research on aspects of modern cell therapy might be qualified as too costly, but will be more targeted and will at least avoid expensive and unjustified clinical studies maximizing

their amounts over time and influences from *in vivo* components on them.

the best use of the available R&D resources for better outcomes.

authorized under the new scheme in 2015 and 2016.

262 Stem Cells in Clinical Practice and Tissue Engineering

expensive novel therapeutic modalities in patient care [98].

possible infections.

**9. Conclusion**

All authors RP, SKP and CS do not have any conflict of interest. HH is the CEO of Zellwerk GmbH.

### **Author details**

Ralf Pörtner1 \*, Shreemanta K. Parida2 , Christiane Schaffer<sup>1</sup> and Hans Hoffmeister<sup>3</sup>

\*Address all correspondence to: poertner@tuhh.de

1 Hamburg University of Technology, Institute of Bioprocess and Biosystems Engineering, Hamburg, Germany

2 Independent Global Health and Translational Medicine Consultant, Berlin, Germany

3Zellwerk GmbH, Oberkrämer, Germany

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**Chapter 13**

**Provisional chapter**

**Tissue Engineering Applications in Maxillofacial**

**Tissue Engineering Applications in Maxillofacial** 

DOI: 10.5772/intechopen.70904

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

and reproduction in any medium, provided the original work is properly cited.

**Keywords:** tissue engineering, scaffolds, bioprinting, stem cells, regenerative medicine,

Nowadays, oral and maxillofacial surgeons face serious difficulties in reconstruction of large defects caused by trauma, cancer, or congenital deformities. Considering each part of oral and maxillofacial region consisting of several tissues, it is necessary to reconstruct these architectures layer by layer. Through years surgeons use different forms of grafts to reconstruct these defects. As these grafts and techniques are well described and used routinely, it should have been noticed that they are not without complications. This is where idea behind tissue engineering steps in. "Tissue engineering" due to its multi-aspect properties can be defined as application of methods and science of engineering toward the understanding of structure-function relationships of mammalian tissues in both normal and pathological forms to improve and develop biologic substitutes to reach the main goal of restoring, maintaining, and stabilization of tissue function. From standpoint of surgery, tissue engineering is not considered as a potential step anymore, but as an available approach to reach the ultimate goal of reconstruction procedures. The aim of this chapter is to defne concepts and advances in tissue engineering (TE). Also, review TE applications in the field of oral and maxillofacial surgery with bolding its clinical applications and complications based on novel and high-qual-

Seied Omid Keyhan, Hamidreza Fallahi,

Seied Omid Keyhan, Hamidreza Fallahi,

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Seyed Mohammad Reza Masoumi, Mohammad Hossein Khosravi and Mohammad Hosein Amirzade-Iranaq

Seyed Mohammad Reza Masoumi, Mohammad Hossein Khosravi and Mohammad Hosein Amirzade-Iranaq

http://dx.doi.org/10.5772/intechopen.70904

ity published researches.

oral surgery, maxillofacial surgery

**Surgery**

**Surgery**

Alireza Jahangirnia,

**Abstract**

Alireza Jahangirnia,


#### **Tissue Engineering Applications in Maxillofacial Surgery Tissue Engineering Applications in Maxillofacial Surgery**

DOI: 10.5772/intechopen.70904

Seied Omid Keyhan, Hamidreza Fallahi, Alireza Jahangirnia, Seyed Mohammad Reza Masoumi, Mohammad Hossein Khosravi and Mohammad Hosein Amirzade-Iranaq Seied Omid Keyhan, Hamidreza Fallahi, Alireza Jahangirnia, Seyed Mohammad Reza Masoumi, Mohammad Hossein Khosravi and Mohammad Hosein Amirzade-Iranaq

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.70904

#### **Abstract**

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[98] Lawler M, French D, Henderson R, Aggarwal A, Sullivan R. Shooting for the moon or flying too near the sun? Crossing the value rubicon in precision cancer care. Public

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Journal of Immunotherapy. 2010;**33**:305-315

[94] ATMP Regulation - LFB CellForCure. 2017. www.cellforcure.com/atmp-regulation/

Nowadays, oral and maxillofacial surgeons face serious difficulties in reconstruction of large defects caused by trauma, cancer, or congenital deformities. Considering each part of oral and maxillofacial region consisting of several tissues, it is necessary to reconstruct these architectures layer by layer. Through years surgeons use different forms of grafts to reconstruct these defects. As these grafts and techniques are well described and used routinely, it should have been noticed that they are not without complications. This is where idea behind tissue engineering steps in. "Tissue engineering" due to its multi-aspect properties can be defined as application of methods and science of engineering toward the understanding of structure-function relationships of mammalian tissues in both normal and pathological forms to improve and develop biologic substitutes to reach the main goal of restoring, maintaining, and stabilization of tissue function. From standpoint of surgery, tissue engineering is not considered as a potential step anymore, but as an available approach to reach the ultimate goal of reconstruction procedures. The aim of this chapter is to defne concepts and advances in tissue engineering (TE). Also, review TE applications in the field of oral and maxillofacial surgery with bolding its clinical applications and complications based on novel and high-quality published researches.

**Keywords:** tissue engineering, scaffolds, bioprinting, stem cells, regenerative medicine, oral surgery, maxillofacial surgery

and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **1. Introduction**

For the first time, Langer and Vacanti introduced the definition of tissue engineering [1] to explain the basics of functional substitutes for tissue damage and how to reconstruct and regenerate these tissues based on principles of biology and medical engineering. This new field in contrast to the former biomaterial thoughts presents incredible disciplines which diverse the goal of regeneration induction of traumatized or damaged tissue rather than substitution with inert parts. In recent decades, a number of articles were being published about the tissue engineering and regenerative medicine (TERM) field over 360 yearly just at the beginning of the twenty-first century. Just in 2010, the number of original articles in this field reaches 4000. This over-increasing attraction to this field—involving almost all tissues even whole organs—leads to researches across the world [2].

that has the potential to create a great impact on the future treatments. One of the major obstacles to the proper functioning of the tissue outside the body is to understand the way in which cells can be set in niches under certain physical and chemical conditions which would be difficult [8, 9]. In this case, bioreactors can control the situation and imitate the natural environment. Bioreactor devices can control and adjust the physiological conditions. With advances in tissue engineering, scaffold design could put several layers of cells onto scaffolds for three-dimensional position. That purpose requires a microenvironment for growth in vitro. The mathematical model was able to calculate the fluid flow rate for scaffold to provide nutrients and remove wastes and release oxygen used. As well as other variables such as external mechanical force needed to stimulate the proliferation of osteoblasts and then followed it can also be provided. Another alternative method that involves the cultivation of graft in vivo using animal models or humans as a bioreactor to simulate the growth of cells is provided. The remainder of this chapter presents various examples discussed regarding

Tissue Engineering Applications in Maxillofacial Surgery http://dx.doi.org/10.5772/intechopen.70904 273

The atrophic mandible presents its own unique set of challenges in reconstructive maxillofacial surgery. A mandibular vertical height of less than 2 cm (20 mm) is universally considered atrophic and presents with characteristic anatomic and physiologic features, such as hypovascularity, which might contribute to tooth and alveolar process loss. The atrophic resorption patterns also contribute to the consistent anatomic changes, such as prominent mylohyoid and internal oblique ridges, which are covered with a thin mucosal lining, contributing to an increased risk of soft tissue breakdown and dehiscence. These anatomic changes happen secondary to a deficiency in blood supply from the lack of muscle attachments in those areas, whereas the areas that have a healthy musculature show an increased blood supply, making it more resistant to postdental extraction resorption. An important concept that reconstructive surgeons need to understand is that atrophic mandibles depend heavily on periosteal

Cawood and his group from the United Kingdom found that alveolar bone resorption seemed

blood supply because of the narrowing of the inferior alveolar artery [10–12].

• Class IV, knife-edge ridge form, adequate in height and inadequate in width

• Class VI, depressed ridge form, with some basilar bone loss evident [13]

• Class III, well-rounded ridge form, adequate in height and width

• Class V, flat ridge form, inadequate in height and width

various tissues of the jaw and face [9].

**3.1. Mandibular defects**

to have a predictable pattern:

• Class II, immediately post-extraction

• Class I, dentate

**3. Oral and maxillofacial bone defects**

Herein, we review latest scientific researches and recent advances of tissue engineering in major field of oral and maxillofacial surgery by subtopics categorized by facial complex parts.

#### **2. Basic principles of tissue engineering**

Tissue engineering is composed of three pillars: the cells, scaffolds, and growth factors. The combination of cells in a suitable scaffold was designed by the appropriate biochemical signals that can facilitate and make possible growth, so it could be a treatment option that is very suitable for clinical application. Various studies have shown that one of the important issues is proper design of scaffolds and associated mechanical signals to regulate tissue that is engineered. Scaffold that can be temporarily or permanently used for three-dimensional porous can also be natural or artificial, which in any case must be biocompatible [3]. A biocompatible environmental issue is crucial importance because it facilitates progenitor cells for migration and differentiation [4]. Some of the important issues that include the physical properties of the scaffold such as biodegradability, porosity, hardness and strength to be as much in excess of migration, cell adhesion, and proliferation (such as osteoconduction), which reflects the influence of signals on the cell is followed by the clinical efficacy of chemical signals and ultimately success factor for the link to be followed. Perhaps the problem for surgeons and maxillofacial surgery is more important than other counterparts, being careful scaffold designing on human anatomy for the repair of any defects in the face. Various studies designed to use the computer in the exact scaffold have shown promising results and have built a biomimetic scaffold that has special significance [5].

To complement these three pillars, tissue, cell lines that require ease of access and availability, differentiation capacity, and lack of stimulation of the immune system or have tumor genesis [6]. Choosing the right cell lines in tissue engineering is still under discussion. New research hopes to use stem cells and gene therapy with viral vectors to express growth factors in cultured cell lines successfully, but stem cell research is outside the scope of this topic [7]. Today, the laboratory of tissue engineering that leads clinicians to living tissue is a concept that has the potential to create a great impact on the future treatments. One of the major obstacles to the proper functioning of the tissue outside the body is to understand the way in which cells can be set in niches under certain physical and chemical conditions which would be difficult [8, 9]. In this case, bioreactors can control the situation and imitate the natural environment. Bioreactor devices can control and adjust the physiological conditions. With advances in tissue engineering, scaffold design could put several layers of cells onto scaffolds for three-dimensional position. That purpose requires a microenvironment for growth in vitro. The mathematical model was able to calculate the fluid flow rate for scaffold to provide nutrients and remove wastes and release oxygen used. As well as other variables such as external mechanical force needed to stimulate the proliferation of osteoblasts and then followed it can also be provided. Another alternative method that involves the cultivation of graft in vivo using animal models or humans as a bioreactor to simulate the growth of cells is provided. The remainder of this chapter presents various examples discussed regarding various tissues of the jaw and face [9].

### **3. Oral and maxillofacial bone defects**

#### **3.1. Mandibular defects**

**1. Introduction**

272 Stem Cells in Clinical Practice and Tissue Engineering

parts.

For the first time, Langer and Vacanti introduced the definition of tissue engineering [1] to explain the basics of functional substitutes for tissue damage and how to reconstruct and regenerate these tissues based on principles of biology and medical engineering. This new field in contrast to the former biomaterial thoughts presents incredible disciplines which diverse the goal of regeneration induction of traumatized or damaged tissue rather than substitution with inert parts. In recent decades, a number of articles were being published about the tissue engineering and regenerative medicine (TERM) field over 360 yearly just at the beginning of the twenty-first century. Just in 2010, the number of original articles in this field reaches 4000. This over-increasing attraction to this field—involving almost all tissues even

Herein, we review latest scientific researches and recent advances of tissue engineering in major field of oral and maxillofacial surgery by subtopics categorized by facial complex

Tissue engineering is composed of three pillars: the cells, scaffolds, and growth factors. The combination of cells in a suitable scaffold was designed by the appropriate biochemical signals that can facilitate and make possible growth, so it could be a treatment option that is very suitable for clinical application. Various studies have shown that one of the important issues is proper design of scaffolds and associated mechanical signals to regulate tissue that is engineered. Scaffold that can be temporarily or permanently used for three-dimensional porous can also be natural or artificial, which in any case must be biocompatible [3]. A biocompatible environmental issue is crucial importance because it facilitates progenitor cells for migration and differentiation [4]. Some of the important issues that include the physical properties of the scaffold such as biodegradability, porosity, hardness and strength to be as much in excess of migration, cell adhesion, and proliferation (such as osteoconduction), which reflects the influence of signals on the cell is followed by the clinical efficacy of chemical signals and ultimately success factor for the link to be followed. Perhaps the problem for surgeons and maxillofacial surgery is more important than other counterparts, being careful scaffold designing on human anatomy for the repair of any defects in the face. Various studies designed to use the computer in the exact scaffold have shown promising

results and have built a biomimetic scaffold that has special significance [5].

To complement these three pillars, tissue, cell lines that require ease of access and availability, differentiation capacity, and lack of stimulation of the immune system or have tumor genesis [6]. Choosing the right cell lines in tissue engineering is still under discussion. New research hopes to use stem cells and gene therapy with viral vectors to express growth factors in cultured cell lines successfully, but stem cell research is outside the scope of this topic [7]. Today, the laboratory of tissue engineering that leads clinicians to living tissue is a concept

whole organs—leads to researches across the world [2].

**2. Basic principles of tissue engineering**

The atrophic mandible presents its own unique set of challenges in reconstructive maxillofacial surgery. A mandibular vertical height of less than 2 cm (20 mm) is universally considered atrophic and presents with characteristic anatomic and physiologic features, such as hypovascularity, which might contribute to tooth and alveolar process loss. The atrophic resorption patterns also contribute to the consistent anatomic changes, such as prominent mylohyoid and internal oblique ridges, which are covered with a thin mucosal lining, contributing to an increased risk of soft tissue breakdown and dehiscence. These anatomic changes happen secondary to a deficiency in blood supply from the lack of muscle attachments in those areas, whereas the areas that have a healthy musculature show an increased blood supply, making it more resistant to postdental extraction resorption. An important concept that reconstructive surgeons need to understand is that atrophic mandibles depend heavily on periosteal blood supply because of the narrowing of the inferior alveolar artery [10–12].

Cawood and his group from the United Kingdom found that alveolar bone resorption seemed to have a predictable pattern:


This classification has more relevance to implant dentistry because it gives the operator an idea of whether an adjunctive bone graft would be necessary (classes IV and V).

A prefabricated titanium mesh was filled with bone mineral blocks, BMPs, and the patient's own bone marrow. Although a clinically successful result was obtained, this procedure may not be as cost-effective as some of the more traditional and established methods of free tissue transfer for mandibular reconstruction and does carry with it significant morbidity related to the surgery itself and potential complications, such as brachial plexus injury and shoulder drop [21]. Nonetheless, it certainly does open up a different aspect of tissue engineering and

Tissue Engineering Applications in Maxillofacial Surgery http://dx.doi.org/10.5772/intechopen.70904 275

It is challenging to reconstruct the edentulous posterior maxilla with dental implants due to insufficient bone height after crestal bone resorption and also maxillary sinus pneumatization [24, 25]. In recent years, with aid of existing space in the maxillary sinus, clinicians introduced techniques for surgical augmentation that use to restore bone height and also create a sufficient implant bed area which seems to resolve patient's treatment difficulties [26, 27]. Researchers suggested a variety of modifications in original sinus augmentation technique to ease different difficulties for clinicians and also patients [28, 29], but the basic principle of each technique remained unchanged which is to increase maxillary bone height with aid of placing graft material in the maxillary sinus after attending to detach the sinus membrane [25, 28, 29]. Nowadays, for rehabilitation of the posterior maxilla with dental implants, the use of maxillary sinus augmentation (MSA) is considered as a standard pro-

In original technique, before dental implant insertion, MSA was performed with the autogenous bone [25, 30]. Autogenous bone has usually been cited as the most eligible material to achieve predictable and favorable results in MSA. It is due to the fact that autogenous bone contains living cells and growth factors which cause osteogenic ability [25, 30]. In contrast, it should have been noticed that available supplies for autogenous bone are limited. Also, as disadvantage, harvesting autogenous bone is painful and includes procedures with risk of infection. With these in mind, it is necessary to investigate and develop alternative techniques

Introduction of different osteoconductive biomaterials such as allogeneic bone [31, 32], xenogeneic bone [32–34], or alloplastic or composite materials [34, 35] which are cell-free and due to that require more time for bone healing. This is a disadvantage that none of mentioned materials have biological and structural properties similar to the native bone [24, 25, 34–37]. The modern science of bone tissue engineering, a fusion of recent discoveries in the field of molecular cell biology with the most innovative methods of reconstructive surgery, aims to

In **Table 1**, studies of stem cell approach for tissue engineering dealing with sinus augmenta-

A major disadvantage of potential bone substitutes is their inherent slow ability to induce new bone at a foreseeable rate. By advances and innovation in technology in tissue engineering, introduced alternative materials which are used as bone show significant advantages in

strategies for maxillofacial reconstruction [22, 23].

to overcome these drawbacks [24, 25, 31–37].

overcome these boundaries [38].

tion were illustrated.

**3.2. Maxillary sinus augmentation**

cedure [24, 25].

Marx and colleagues [14] published a novel soft tissue matrix expansion also known as the "tent pole," where the dental implants effectively "tent" the soft tissue envelope up to maintain the bone graft volume and prevent soft tissue collapse. The original description used an extraoral submental approach, and the bone graft material of choice was the anterior iliac crest bone graft, with four to five implants placed (each one 15 mm in height), with a 1-cm interimplant distance. Primary stability was obtained by engaging the inferior border of the mandible with the implants. Autogenous corticocancellous bone graft is then packed around the implants.

Patel et al. reported that the addition of rhBMP in the tent pole technique had a favorable impact on bone healing and allowed substitution of the posterior iliac crest as a donor site with the anterior iliac crest bone graft because of the enhanced osteoinduction that happens with rhBMP. Furthermore, the authors rarely use the classical anterior iliac crest bone grafting approach, instead opting for the trephine to harvest the bone from the anterior iliac crest, with excellent increase in vertical bone height and final implant placement. This translated to less donor-site morbidity and earlier mobilization [15].

Many surgeons have modified Marx's original tent pole technique, and some have replaced dental implants with bone screws; this modification seemed to improve the buccolingual orientation of the final implant placement, because the dental implants would be placed at a second procedure, when all of the bones have been consolidated, and the position of the implants is more ideal. A second advantage of this modification is that it allows the use of surgical implant guides, especially if a maxillary prosthesis exists. Another commonly used method is the use of a titanium mesh to tent the soft tissue and maintain the bone graft and the contour of the ridge. However, the main disadvantage of this technique is that the surgical site must be reentered to remove the titanium mesh before implant placement. This has presented its own set of challenges, especially when the graft grows over the mesh, and the procedure requires excessive soft tissue reflection.

Another bioactive agent that has been studied in maxillofacial reconstructive surgery is recombinant human platelet-derived growth factor. This is a product of platelets and functions as a chemotactic and mitogenic factor for osteoblasts and is critical for angiogenesis and thus can be applied to treating ridge defects [16]. This growth factor has been combined with several different types of grafting materials and carriers, such as mineralized and demineralized FDBA [17], xenograft (specifically deproteinized bovine block graft), equine block graft [18], and bTCP [19], in multiple case series and has been shown to help produce intact woven and lamellar bone contributing to an increase in vertical ridge height in humans, which was of appropriate quality to accommodate the placement of dental implants at a second stage. The concept of engineered heterotopic bone formation has also been studied; however, this has not yet gained much notoriety. In 2004, it was studied in the reconstruction of large segmental mandibular defects by way of an engineered growth of a mandibular transplant within a muscular environment (in this case the latissimus dorsi muscle) with the help of BMPs, with subsequent free tissue transfer of the bone-muscle flap approximately 7 weeks later [20]. A prefabricated titanium mesh was filled with bone mineral blocks, BMPs, and the patient's own bone marrow. Although a clinically successful result was obtained, this procedure may not be as cost-effective as some of the more traditional and established methods of free tissue transfer for mandibular reconstruction and does carry with it significant morbidity related to the surgery itself and potential complications, such as brachial plexus injury and shoulder drop [21]. Nonetheless, it certainly does open up a different aspect of tissue engineering and strategies for maxillofacial reconstruction [22, 23].

#### **3.2. Maxillary sinus augmentation**

This classification has more relevance to implant dentistry because it gives the operator an

Marx and colleagues [14] published a novel soft tissue matrix expansion also known as the "tent pole," where the dental implants effectively "tent" the soft tissue envelope up to maintain the bone graft volume and prevent soft tissue collapse. The original description used an extraoral submental approach, and the bone graft material of choice was the anterior iliac crest bone graft, with four to five implants placed (each one 15 mm in height), with a 1-cm interimplant distance. Primary stability was obtained by engaging the inferior border of the mandible with the implants. Autogenous corticocancellous bone graft is then packed around

Patel et al. reported that the addition of rhBMP in the tent pole technique had a favorable impact on bone healing and allowed substitution of the posterior iliac crest as a donor site with the anterior iliac crest bone graft because of the enhanced osteoinduction that happens with rhBMP. Furthermore, the authors rarely use the classical anterior iliac crest bone grafting approach, instead opting for the trephine to harvest the bone from the anterior iliac crest, with excellent increase in vertical bone height and final implant placement. This translated to less

Many surgeons have modified Marx's original tent pole technique, and some have replaced dental implants with bone screws; this modification seemed to improve the buccolingual orientation of the final implant placement, because the dental implants would be placed at a second procedure, when all of the bones have been consolidated, and the position of the implants is more ideal. A second advantage of this modification is that it allows the use of surgical implant guides, especially if a maxillary prosthesis exists. Another commonly used method is the use of a titanium mesh to tent the soft tissue and maintain the bone graft and the contour of the ridge. However, the main disadvantage of this technique is that the surgical site must be reentered to remove the titanium mesh before implant placement. This has presented its own set of challenges, especially when the graft grows over the mesh, and the procedure requires

Another bioactive agent that has been studied in maxillofacial reconstructive surgery is recombinant human platelet-derived growth factor. This is a product of platelets and functions as a chemotactic and mitogenic factor for osteoblasts and is critical for angiogenesis and thus can be applied to treating ridge defects [16]. This growth factor has been combined with several different types of grafting materials and carriers, such as mineralized and demineralized FDBA [17], xenograft (specifically deproteinized bovine block graft), equine block graft [18], and bTCP [19], in multiple case series and has been shown to help produce intact woven and lamellar bone contributing to an increase in vertical ridge height in humans, which was of appropriate quality to accommodate the placement of dental implants at a second stage. The concept of engineered heterotopic bone formation has also been studied; however, this has not yet gained much notoriety. In 2004, it was studied in the reconstruction of large segmental mandibular defects by way of an engineered growth of a mandibular transplant within a muscular environment (in this case the latissimus dorsi muscle) with the help of BMPs, with subsequent free tissue transfer of the bone-muscle flap approximately 7 weeks later [20].

idea of whether an adjunctive bone graft would be necessary (classes IV and V).

the implants.

274 Stem Cells in Clinical Practice and Tissue Engineering

donor-site morbidity and earlier mobilization [15].

excessive soft tissue reflection.

It is challenging to reconstruct the edentulous posterior maxilla with dental implants due to insufficient bone height after crestal bone resorption and also maxillary sinus pneumatization [24, 25]. In recent years, with aid of existing space in the maxillary sinus, clinicians introduced techniques for surgical augmentation that use to restore bone height and also create a sufficient implant bed area which seems to resolve patient's treatment difficulties [26, 27]. Researchers suggested a variety of modifications in original sinus augmentation technique to ease different difficulties for clinicians and also patients [28, 29], but the basic principle of each technique remained unchanged which is to increase maxillary bone height with aid of placing graft material in the maxillary sinus after attending to detach the sinus membrane [25, 28, 29]. Nowadays, for rehabilitation of the posterior maxilla with dental implants, the use of maxillary sinus augmentation (MSA) is considered as a standard procedure [24, 25].

In original technique, before dental implant insertion, MSA was performed with the autogenous bone [25, 30]. Autogenous bone has usually been cited as the most eligible material to achieve predictable and favorable results in MSA. It is due to the fact that autogenous bone contains living cells and growth factors which cause osteogenic ability [25, 30]. In contrast, it should have been noticed that available supplies for autogenous bone are limited. Also, as disadvantage, harvesting autogenous bone is painful and includes procedures with risk of infection. With these in mind, it is necessary to investigate and develop alternative techniques to overcome these drawbacks [24, 25, 31–37].

Introduction of different osteoconductive biomaterials such as allogeneic bone [31, 32], xenogeneic bone [32–34], or alloplastic or composite materials [34, 35] which are cell-free and due to that require more time for bone healing. This is a disadvantage that none of mentioned materials have biological and structural properties similar to the native bone [24, 25, 34–37].

The modern science of bone tissue engineering, a fusion of recent discoveries in the field of molecular cell biology with the most innovative methods of reconstructive surgery, aims to overcome these boundaries [38].

In **Table 1**, studies of stem cell approach for tissue engineering dealing with sinus augmentation were illustrated.

A major disadvantage of potential bone substitutes is their inherent slow ability to induce new bone at a foreseeable rate. By advances and innovation in technology in tissue engineering, introduced alternative materials which are used as bone show significant advantages in


their bone-inductive capabilities. These results are similar to the result of study conducted by Neiva et al., which revealed favorable outcomes with the use of PepGen P-15 Putty. They conclude this result from initial osteogenesis of intervened group which is guided by the putty and achieved an additional mature trabecular pattern in shorter period of time comparing to the control group. Earlier bone formation is evaluated and revealed in 3D radiographic

**Study Control Type SC Scaffold Evaluation Follow-up Complications**

BBM + polymer fleece

BBM, bovine bone mineral; HA/TCP, hydroxyapatite/tricalcium phosphate; NA, data not available; MSCs, mesenchymal

D: 13 of 14 sinuses showed insufficient bone formation. Resorption 90% in test group after 3 months, 29% in control

B: Histology shows little or no bone formation in 8 of 15 patients, when a two-stage protocol was used. C: Less bone and more medullar spaces were found in test group compared with control group.

Histology after 4 and 6 months

18 months None

277

Tissue Engineering Applications in Maxillofacial Surgery http://dx.doi.org/10.5772/intechopen.70904

A variety of study designs used in cellular studies on sinus grafting techniques outcome the square measure constituent. Briefly, most studies comparing cell therapy with a traditional

As application of allografts for dorsal augmentation seems to have serious disadvantages [45], it appears that we might tend to observe the appliance of tissue engineering in rhinoplasty; Kim et al. in 2014 within the article made a case for the chondrocytes and porcine cartilage substance (PCS) construct as an attainable dorsal augmentation material in rhinoplasty cultured with a porcine cartilage-derived substance (PCS) scaffold as a potential substitute for normal tissue use for augmentation in rhinoplasty. A scaffold is derived from decellularized and fine-grained porcine articular cartilage prepared. The use of the rabbit articular cartilage was due to ability to supply homologous chondrocytes, which for 7 weeks were enlarged and polite with the PCS scaffold. The chondrocyte-PCS constructs were then surgically implanted on the nasal dorsum of six rabbits. Four and 8 weeks after implantation, complete evaluations such as the gross morphology, radiologic pictures, and microscopic anatomy options of the location of implant were analyzed. The rabbits showed no signs of surgical inflammation and infection. The degree of dorsal augmentation was maintained throughout the 8-week surgical observation amount. Surgical examinations showed chondrocyte proliferation while there was no inflammatory response. However, neo-cartilage formation from the constructs was not confirmed. The biocompatibility and structural options of tissue-engineered chondrocyte-PCS constructs indicate their potential as candidate dorsal augmentation material to be used

assessment as early as 8 weeks [40].

None MSC

**Table 1.** Studies regarding cell-based sinus lift procedure.

Adopted from Jakobsen et al. [39]

(periosteum)

stem cells; PRP, platelet-rich plasma; RCT, randomized clinical trial A: 11 augmentations failed in test group, none in control group

Beaumont et al.

group

grafting technique showed similar results [41–44].

**3.3. Dorsal augmentation in rhinoplasty**

in rhinoplasty [46].


Adopted from Jakobsen et al. [39]

**Study Control Type SC Scaffold Evaluation Follow-up Complications**

Histology after 3–4 months

histology on 18 patients

3 months

13–16 weeks

3 months

Histology after 4 months

6 months, CT scan

after 3 months

3 months

6 months

6 months

4 months

Histology after 4 months

3–4 months

Histology after 6–8 months

PRP Radiography 2–5 years None

PRP Radiography 2- to

Polymer fleece Radiography,

HA/TCP Histology after

BBM Histology after

Polymer fleece Histology after

Polymer fleece Histology after

Polymer fleece Radiography

BBM Histology after

BBM Histology after

Polymer fleece Histology after

Polymer fleece Histology after

BBM Histology after

50% ABG + 50%

Collagen [1] BBM (2a)

BBM

None

None

None

B

C

D

None

None

None

None

12 months None

5 years None

8 months None

None

24 months A

12 months None

6.3-year

6 months in 9 patients

Allograft (Osteocel)

Allograft (Osteocel)

Gonshor et al. Allograft MSC (bone

276 Stem Cells in Clinical Practice and Tissue Engineering

Shayesteh et al. None MSC (bone

Yamada et al. None MSC (bone

and 70% BBM

phosphate

Ueda et al. None MSC (bone

marrow) vs. BMAC

Fuerst et al. None Autogenous

None MSC

None MSC

ABG + 50% BBM

ABG + 70% BBM

Springer et al. BBM MSC

None MSC

ABG MSC

None MSC (bone

Rickert et al. 30% ABG

Mangano et al. Calcium

Sauerbier et al. MSC (bone

Schimming et al.

MacAllister et al.

Zizelmann et al.

Trautvetter et al.

Schmelziesen et al.

Hermund et al. 50%

Sauerbier et al. 30%

Voss et al. ABG MSC

marrow)

marrow)

marrow)

MSC (bone marrow)

(periosteum)

marrow)

MSC (bone marrow)

(periosteum)

marrow)

MSC (bone marrow)

culture-expanded bone cells

(periosteum)

(periosteum)

Autogenous culture-expanded bone cells

MSC (bone marrow)

(periosteum) (l) Autogenous culture-expanded bone cells (2a)

(periosteum)

BBM, bovine bone mineral; HA/TCP, hydroxyapatite/tricalcium phosphate; NA, data not available; MSCs, mesenchymal stem cells; PRP, platelet-rich plasma; RCT, randomized clinical trial

A: 11 augmentations failed in test group, none in control group

B: Histology shows little or no bone formation in 8 of 15 patients, when a two-stage protocol was used.

C: Less bone and more medullar spaces were found in test group compared with control group.

D: 13 of 14 sinuses showed insufficient bone formation. Resorption 90% in test group after 3 months, 29% in control group

**Table 1.** Studies regarding cell-based sinus lift procedure.

their bone-inductive capabilities. These results are similar to the result of study conducted by Neiva et al., which revealed favorable outcomes with the use of PepGen P-15 Putty. They conclude this result from initial osteogenesis of intervened group which is guided by the putty and achieved an additional mature trabecular pattern in shorter period of time comparing to the control group. Earlier bone formation is evaluated and revealed in 3D radiographic assessment as early as 8 weeks [40].

A variety of study designs used in cellular studies on sinus grafting techniques outcome the square measure constituent. Briefly, most studies comparing cell therapy with a traditional grafting technique showed similar results [41–44].

#### **3.3. Dorsal augmentation in rhinoplasty**

As application of allografts for dorsal augmentation seems to have serious disadvantages [45], it appears that we might tend to observe the appliance of tissue engineering in rhinoplasty; Kim et al. in 2014 within the article made a case for the chondrocytes and porcine cartilage substance (PCS) construct as an attainable dorsal augmentation material in rhinoplasty cultured with a porcine cartilage-derived substance (PCS) scaffold as a potential substitute for normal tissue use for augmentation in rhinoplasty. A scaffold is derived from decellularized and fine-grained porcine articular cartilage prepared. The use of the rabbit articular cartilage was due to ability to supply homologous chondrocytes, which for 7 weeks were enlarged and polite with the PCS scaffold. The chondrocyte-PCS constructs were then surgically implanted on the nasal dorsum of six rabbits. Four and 8 weeks after implantation, complete evaluations such as the gross morphology, radiologic pictures, and microscopic anatomy options of the location of implant were analyzed. The rabbits showed no signs of surgical inflammation and infection. The degree of dorsal augmentation was maintained throughout the 8-week surgical observation amount. Surgical examinations showed chondrocyte proliferation while there was no inflammatory response. However, neo-cartilage formation from the constructs was not confirmed. The biocompatibility and structural options of tissue-engineered chondrocyte-PCS constructs indicate their potential as candidate dorsal augmentation material to be used in rhinoplasty [46].

Cultured chondrocytes and porcine cartilage substance (PCS) constructs as an attainable dorsal augmentation material in rhinoplasty: preliminary animal study and, additionally Mendelson et al. in 2014 conferred this concept than designed nasal cartilage by cell homing: a model for augmentative and rehabilitative rhinoplasty. Bioactive scaffolds were developed that not solely recruited cells within the nasal dorsum in vivo, however, additionally induced chondrogenesis of the recruited cells. Bilayered scaffolds were fictional with alginate-containing gelatin microspheres encapsulating cytokines atop a porous poly(lactic-co-glycolic acid) base. Microspheres were fictional to contain recombinant human remodeling growth factor β3 at doses of 200, 500, or 1000 ng, with phosphate-buffered saline-loaded microspheres used as a bearing. A rat model of augmentation facelift was created by implanting scaffolds atop the native nasal cartilage surface that was scored to induce cell migration. Tissue formation and chondrogenesis within the scaffolds were evaluated by image analysis and microscopic anatomy staining with hematoxylin and eosin, toluidine blue, Verhoeff elastic-van Gieson, and aggrecan immunohistochemistry. Sustained release of increasing doses of remodeling growth factor β3 for up to the tested 10 weeks promoted orthotopic cartilage-like tissue formation in an exceedingly dose-dependent manner. It appears that these findings represent the primary commitment to engineer cartilage tissue by the cell orientating for facelift and will doubtlessly function as an alternate material for augmentative and rehabilitative rhinoplasty [46].

we believe that these efforts facilitate the way for the new generation of artificial dermal substitute, resulting in the "skin dermis tissue engineering" [51]. Fortunately, after 6 years, both of these groups independently revealed the clinical effect of tissue-engineered substitutes for the treatment of different grade burns, albeit in different approaches. The first graft of extensive burns with sheets of cultured epithelium (produced from epidermal cells which are autologous) was reported by O'Connor et al., two adult patients were experienced at the Peter Bent Brigham Hospital [52, 53]. Cultured epidermal autografts (CEA) were the next generation of cultured sheets which are autologous and also successively revealed to prepare cover of full-thickness burns in pediatric patients [54]. Meantime, only a short time after O'Connor et al.'s study, Burke et al. revealed that artificial dermis had experienced functional and physiological acceptable dermis in the treatment of extensive full-thickness burns on several patients [55]. These evidences resulted in randomized clinical trial for extensive burn injuries led by Heimbach et al. [56] about the application of artificial dermis; now new generation of artificial dermis is known as IntegraTM Dermal Regeneration Template. This study was done successfully by collaborating 11 centers, and other studies [57, 58] could inevitably demonstrate this dermal substitute a "gold standard" material treatment of full-

But there are still challenges, and those two groups are still far from reaching the final goal of replacing autologous skin for coverage of permanent deep dermal or full-thickness injuries

**Table 2** demonstrates the current status of available tissue-engineered materials and tech-

Confluent autologous keratinocytes

**Structure Advantage Disadvantage**

CUONO's method Extensive bums Two-stage procedure,

In vitro expansion for large bum area, permanent

Expansion 1:4, no rejection

Expansion 1:9–15, no rejection, high take rate, shorter epithelization time

Fragility, infection, high cost, and variable take

precise grafting time coordination

Beyond 1:4 expansion: poor cosmetic and functional results, delayed reepithelialization

Time-consuming, laborintensive, hypertrophic

scarring

rate

Tissue Engineering Applications in Maxillofacial Surgery http://dx.doi.org/10.5772/intechopen.70904 279

thickness burns [59].

in extensive burns.

niques for skin substitution.

Epidermal Cultured epithelial

**Skin substitute/ surgical technique**

autograft (CEA)

CEA with meshed split-thickness skin autograft

CEA with microskin autograft

An important feature for rehabilitative and augmentative rhinoplasty is the ability of the graft to be tailored to the individual patient. Autologous graft area unit is stacked and sutured along in a very bundle before implantation. The bioactive poly(lactic-co-glycolic acid) scaffolds are simply changed to larger augmentations by varied the mold diameter wont to create the poly(lactic-co-glycolic acid) scaffold base. For associate degree off-the-shelf product, three totally different scaffolds can be generated with a variety of forms and sizes and simply cut for precise adjustments. Thus, the bioactive scaffolds might probably be used as completely unique various implant styles to current rhinoplasty treatment [46].

### **4. Skin**

It seems that one of the important years for tissue engineering is 1975; in this year, some occurrence about skin engineering was evolved in this field even though the Washington National Science Foundation applied science panel meeting to formally adopt the term "tissue engineering" for this field a decade later in 1987 [47] and Langer and Vacanti explained the definition of this field later in 1993 [1]. The first step is ascribed to the actions of two teams in the United States 40 years ago. Rheinwatd and Green were the first team who are unskilled and ignored cultivation of human epidermal keratinocytes in vitro [48]; they also created potentially the enlargement of those cultivation of cells into numerous epithelial cells for graft in 1975 [49] from a little skin diagnostic assay. Today, the work that was done in those days is called "skin epidermis tissue engineering." At the same time, Yannas et al. worked on the features of scleroprotein and degradation mechanism [50] in 1975, which now we believe that these efforts facilitate the way for the new generation of artificial dermal substitute, resulting in the "skin dermis tissue engineering" [51]. Fortunately, after 6 years, both of these groups independently revealed the clinical effect of tissue-engineered substitutes for the treatment of different grade burns, albeit in different approaches. The first graft of extensive burns with sheets of cultured epithelium (produced from epidermal cells which are autologous) was reported by O'Connor et al., two adult patients were experienced at the Peter Bent Brigham Hospital [52, 53]. Cultured epidermal autografts (CEA) were the next generation of cultured sheets which are autologous and also successively revealed to prepare cover of full-thickness burns in pediatric patients [54]. Meantime, only a short time after O'Connor et al.'s study, Burke et al. revealed that artificial dermis had experienced functional and physiological acceptable dermis in the treatment of extensive full-thickness burns on several patients [55]. These evidences resulted in randomized clinical trial for extensive burn injuries led by Heimbach et al. [56] about the application of artificial dermis; now new generation of artificial dermis is known as IntegraTM Dermal Regeneration Template. This study was done successfully by collaborating 11 centers, and other studies [57, 58] could inevitably demonstrate this dermal substitute a "gold standard" material treatment of fullthickness burns [59].

Cultured chondrocytes and porcine cartilage substance (PCS) constructs as an attainable dorsal augmentation material in rhinoplasty: preliminary animal study and, additionally Mendelson et al. in 2014 conferred this concept than designed nasal cartilage by cell homing: a model for augmentative and rehabilitative rhinoplasty. Bioactive scaffolds were developed that not solely recruited cells within the nasal dorsum in vivo, however, additionally induced chondrogenesis of the recruited cells. Bilayered scaffolds were fictional with alginate-containing gelatin microspheres encapsulating cytokines atop a porous poly(lactic-co-glycolic acid) base. Microspheres were fictional to contain recombinant human remodeling growth factor β3 at doses of 200, 500, or 1000 ng, with phosphate-buffered saline-loaded microspheres used as a bearing. A rat model of augmentation facelift was created by implanting scaffolds atop the native nasal cartilage surface that was scored to induce cell migration. Tissue formation and chondrogenesis within the scaffolds were evaluated by image analysis and microscopic anatomy staining with hematoxylin and eosin, toluidine blue, Verhoeff elastic-van Gieson, and aggrecan immunohistochemistry. Sustained release of increasing doses of remodeling growth factor β3 for up to the tested 10 weeks promoted orthotopic cartilage-like tissue formation in an exceedingly dose-dependent manner. It appears that these findings represent the primary commitment to engineer cartilage tissue by the cell orientating for facelift and will doubtlessly function as an alternate material for augmentative and rehabilitative rhino-

An important feature for rehabilitative and augmentative rhinoplasty is the ability of the graft to be tailored to the individual patient. Autologous graft area unit is stacked and sutured along in a very bundle before implantation. The bioactive poly(lactic-co-glycolic acid) scaffolds are simply changed to larger augmentations by varied the mold diameter wont to create the poly(lactic-co-glycolic acid) scaffold base. For associate degree off-the-shelf product, three totally different scaffolds can be generated with a variety of forms and sizes and simply cut for precise adjustments. Thus, the bioactive scaffolds might probably be used as completely

It seems that one of the important years for tissue engineering is 1975; in this year, some occurrence about skin engineering was evolved in this field even though the Washington National Science Foundation applied science panel meeting to formally adopt the term "tissue engineering" for this field a decade later in 1987 [47] and Langer and Vacanti explained the definition of this field later in 1993 [1]. The first step is ascribed to the actions of two teams in the United States 40 years ago. Rheinwatd and Green were the first team who are unskilled and ignored cultivation of human epidermal keratinocytes in vitro [48]; they also created potentially the enlargement of those cultivation of cells into numerous epithelial cells for graft in 1975 [49] from a little skin diagnostic assay. Today, the work that was done in those days is called "skin epidermis tissue engineering." At the same time, Yannas et al. worked on the features of scleroprotein and degradation mechanism [50] in 1975, which now

unique various implant styles to current rhinoplasty treatment [46].

plasty [46].

278 Stem Cells in Clinical Practice and Tissue Engineering

**4. Skin**

But there are still challenges, and those two groups are still far from reaching the final goal of replacing autologous skin for coverage of permanent deep dermal or full-thickness injuries in extensive burns.


**Table 2** demonstrates the current status of available tissue-engineered materials and techniques for skin substitution.


The happen which facilitates efforts is that combination between a skin allograft bank and professional laboratory which culture autologous epithelial cell sheet could be an important step, and also we should gather many scientists and engineers and absorb finance in this field. The only way that can create the demand of engineered tissue for patients is through working and collaborating with clinicians, and also this expert team brings us innovation, novel technologies, and cost management and realizes the challenges in advancement of skin tissue engineering [61–65].

glycosaminoglycan substrates containing autologous fibroblasts and

collagen type 1 hydrogels engineered with human keratinocytes and fibroblasts

keratinocytes

DenovoSkin Plastically compressed

**Table 2.** Tissue-engineered materials and current surgical techniques for skin substitution.

**Structure Advantage Disadvantage**

Permanent replacement of both dermal and epidermal layers, one-step procedure

Near-normal skin architecture No clinical trial reported

281

Long culture time, no clinical series reported

yet

Tissue Engineering Applications in Maxillofacial Surgery http://dx.doi.org/10.5772/intechopen.70904

yet

There is a recognized lot to reconstruct and restore advanced craniomaxillofacial (CMF) soft tissues that are broken and/or disfigured as a consequence of automobile accident, trauma, burn injury, or tumor surgery. In trauma, injuries usually produce extraordinarily advanced geometric and avulsion defects, and also the anatomic and purposeful intricacies of CMF composite soft tissue structures like the lips, eyelids, and nasal advanced create the recon-

Kenji Izumi et al. in 2013 within the article evaluated the appliance of tissue engineering in oral mucosa [67]; the first objective of this study was to gauge the security of a tissueengineered human ex vivo produced oral mucosa equivalent (EVPOME) in intraoral graft procedures. The secondary objective was to assess the efficacy of the grafted EVPOME in manufacturing a keratinized mucosal surface epithelial tissue. Five patients World Health Organization based on inclusion criteria that defects in mucogingival region or an absence of gingiva which is keratinized on incisors and premolars teeth, together with radiographies of adequate bone height in interdental region, were used to expand the amount of keratinized gingiva in the defect site. A specimen was taken by a punch biopsy from hard palate to accumulate oral keratinocytes, which were enlarged, associate degreed cultured on associate

struction significantly difficult for maxillofacial surgeons (**Table 3**).

degree noncellular matrix of the dermis for make of an EVPOME.

**5. Oral mucosa**

Adopted from Chua et al. [60]

**Skin substitute/ surgical technique**

Dermo-epidermal PermaDerm™ Collagen-


**Table 2.** Tissue-engineered materials and current surgical techniques for skin substitution.

The happen which facilitates efforts is that combination between a skin allograft bank and professional laboratory which culture autologous epithelial cell sheet could be an important step, and also we should gather many scientists and engineers and absorb finance in this field. The only way that can create the demand of engineered tissue for patients is through working and collaborating with clinicians, and also this expert team brings us innovation, novel technologies, and cost management and realizes the challenges in advancement of skin tissue engineering [61–65].

### **5. Oral mucosa**

**Skin substitute/ surgical technique**

280 Stem Cells in Clinical Practice and Tissue Engineering

Integra™ with CEA

Integra™ with Meek

Composite skin substitute

AlloDerm® with

CEA

Integra™ Cross-linked bovine

MatriDerm® Bovine non-cross-linked

Blobrane® Silicone membrane and

AlloDerm® Human acellular

Permacol™ Porcine acellular

TransCyte® Porcine collagen-coated

Dermagraft® Bioabsorbable polyglactin

fibroblasts

collagen

lyophilized dermis, coated with alpha-elastin

MatriDerm as a template, seeded with expanded autologous skin fibroblast and keratinocytes

nylon mesh impregnated with porcine dermal

lyophilized dermis

lyophilized dermis

nylon mesh seeded with allogeneic neonatal human foreskin fibroblasts

mesh scaffold seeded with cryopreserved allogeneic neonatal human foreskin

hydrolysate

tendon collagen-based dermal matrix linked with glycosaminoglycan (GAG)

Dermal Artificial biological materials

> Natural biological

Synthetic materials

**Structure Advantage Disadvantage**

Good longterm esthetic and functional outcome

Full wound closure

One-stage procedure, coverage of partial-thickness

bums

bed

Good esthetic and functional

Immediate availability, ease of storage

Ease of handling, no rejection, chronic wounds diabetic ulcers

Acellular, immunologically inert, provide natural dermal porosities for regeneration and vascularization on the wound

Two-stage procedure, infection, hematomas,

High cost, poor adhesion

seromas

Intolerant to

High cost, risk of transmitting disease, two-stage procedure

Multiple applications

Infection, hematomas,

Poor ECM structure, infections, cellulitis,

seromas

Temporary

contaminated wound bed

There is a recognized lot to reconstruct and restore advanced craniomaxillofacial (CMF) soft tissues that are broken and/or disfigured as a consequence of automobile accident, trauma, burn injury, or tumor surgery. In trauma, injuries usually produce extraordinarily advanced geometric and avulsion defects, and also the anatomic and purposeful intricacies of CMF composite soft tissue structures like the lips, eyelids, and nasal advanced create the reconstruction significantly difficult for maxillofacial surgeons (**Table 3**).

Kenji Izumi et al. in 2013 within the article evaluated the appliance of tissue engineering in oral mucosa [67]; the first objective of this study was to gauge the security of a tissueengineered human ex vivo produced oral mucosa equivalent (EVPOME) in intraoral graft procedures. The secondary objective was to assess the efficacy of the grafted EVPOME in manufacturing a keratinized mucosal surface epithelial tissue. Five patients World Health Organization based on inclusion criteria that defects in mucogingival region or an absence of gingiva which is keratinized on incisors and premolars teeth, together with radiographies of adequate bone height in interdental region, were used to expand the amount of keratinized gingiva in the defect site. A specimen was taken by a punch biopsy from hard palate to accumulate oral keratinocytes, which were enlarged, associate degreed cultured on associate degree noncellular matrix of the dermis for make of an EVPOME.


Another researchers believed that the long time (more than 22 months) existence of the cultured keratinocytes from oral cavity, expressed markers of stem cell. Also evidence tried to by pharmacologic approach produce cultured oral keratinocytes to produce stem cell population; and now all efforts focus on clinical application of this technology that has a lot of simply for the event of a lot of strong EVPOME for intraoral graft procedures [75]. As a result of oral mucosa keratinocyte area unit simply getable and expand quicker in vitro than skin keratinocytes [76], they will be a lot of efficacious to be used in future clinical applications in regenerative medicine. This platform technique might produce other potential extraoral uses, like repair of facial skin [77], reconstruction of eyelids and nose, or in situ tissue layer substi-

Tissue Engineering Applications in Maxillofacial Surgery http://dx.doi.org/10.5772/intechopen.70904 283

The temporomandibular joint (TMJ) may be a synovial joint that has for articulator motion relative to the os base and distributes the traditional stresses of perform (chewing and speaking) and parafunction (clenching and bruxism). It is usually noted as ginglymoarthrodial joint attributable to its slippy performance and hinging. The temporomandibular joint links the condyloid process (mandibular bone) to the temporal bone. The cartilage disc is the middle of mandibular condyle and the glenoid fossa eminence of the temporal bone and separates the joint area into inferior and superior compartments, each of that area unit crammed with

Because of the advanced loading patterns that designed tissues can expertise within the TMJ, acquisition of complete style parameters from the native tissue is important. Significantly, TMJ disc, condyle, and condylar cartilage replacement area unit in nice demand attributable to these tissues' poor regenerative capability and high rate of involvement in TMD. In response, many studies characterizing the properties of those elements are performed [79]. Though glenoid fossa and articular eminence are the concern in TMD, they need not be absolutely characterized. Within the following section, structural characteristics of the TMJ tissue area unit are summarized. Application of tissue engineering in the treatment of temporomandibular joint defects is rising as a progressive choice to substitute and remove the pathological defects automatically in the near future. Historically, basics of regenerative medicine are three necessary elements, cells, stimulator factors, and scaffolds. But new technology introduced novel methodologies and recommends how to manage TMJ disk and stimulate relevant cartilage [80]. This approach includes the scaffold-free and cell-based methodology and cells and stimulators that work together. But another approach is to construct a structure and render appropriate form of engineered tissues, permitting well-designed structure and simple handling [81]. Another mechanical feature may be appropriate for designed tissue. In the best manner the chemistry of scaffold that could be degradation with matrix synthesis. But the rate of scaffold degradation depends on the nature of scaffold and might be modified by manipulation. For induction of the mass of matrix synthesis, growth factors are added to the scaffolds. Today, all engineered tissue materials are used to regenerate condyle and TMJ disc; however, similar strategies for regeneration of mandibular fossa are not successful [82].

tutes for the urethra and conjunctiva [78].

synovial fluid [79].

**6. Temporomandibular joint disorders**

**Table 3.** The advantages and downsides of the contemporary approach to soft tissue reconstruction [66].

EVPOME grafts have special features which are used directly over associate degree healthy periosteal bed and preserved in situ. At 1 and at 7, 14, 30, 90, and 180 days postsurgery, Plaque Index and Gingival Index were recorded for every subject. Additionally, inquisitory depths, keratinized animal tissue dimension, and keratinized animal tissue thickness were recorded at baseline, 30, 90, and 180 days. Fortunately, there were no adverse outcomes or complications to EVPOME ascertained in all cases throughout the research. But the mean increased in keratinized animal tissue dimension was 3 mm (range, 3–4 mm), and no vital changes in depths were ascertained. According to our findings, we can terminate that EVPOME is useful for oral application and has the flexibility to reinforce keratinized gingiva. More randomized clinical trials in this field should be performed to demonstrate other dimensions of tissue engineering [67].

The maintenance of associate degree sufficient strip of attached gingiva includes a minimum of 2 mm of keratinized gingiva and has revealed that it could be necessary for preservation of periodontal tissue [68, 69]. Historically, FGGs' associate degreed grafts which were taken from connective tissue are applied to gain sufficient strip of attached gingiva [70]. Unfortunately, clinical evidence about the use of autologous and allogenic products confronts to some problems about FGGs, i.e., problems about morbidity around donor region and also amount of tissue for graft which is restricted [71]. Nevins [72] applied treatment by bilayered cell for mucogingival region, and this research revealed that adequate keratinized tissue was gained; however, the amount was not more than it gained with FGGs. Interestingly, each research tried to prove that clinical evidence such as texture and color have better results when compared with FGGs. Nevertheless, application of products that have allogenic cells could have important effect on the wound bed; the impact of treatment could be as a completely unique "biologic dressing" to motivate encompassing cells. One of the reliable materials was a scaffold which is biodegradable with gingival autologous fibroblasts which are cultured [73]. Another research in this field used oral mucosa cells to transplant directly to the cornea [74]. Another researchers believed that the long time (more than 22 months) existence of the cultured keratinocytes from oral cavity, expressed markers of stem cell. Also evidence tried to by pharmacologic approach produce cultured oral keratinocytes to produce stem cell population; and now all efforts focus on clinical application of this technology that has a lot of simply for the event of a lot of strong EVPOME for intraoral graft procedures [75]. As a result of oral mucosa keratinocyte area unit simply getable and expand quicker in vitro than skin keratinocytes [76], they will be a lot of efficacious to be used in future clinical applications in regenerative medicine. This platform technique might produce other potential extraoral uses, like repair of facial skin [77], reconstruction of eyelids and nose, or in situ tissue layer substitutes for the urethra and conjunctiva [78].

### **6. Temporomandibular joint disorders**

EVPOME grafts have special features which are used directly over associate degree healthy periosteal bed and preserved in situ. At 1 and at 7, 14, 30, 90, and 180 days postsurgery, Plaque Index and Gingival Index were recorded for every subject. Additionally, inquisitory depths, keratinized animal tissue dimension, and keratinized animal tissue thickness were recorded at baseline, 30, 90, and 180 days. Fortunately, there were no adverse outcomes or complications to EVPOME ascertained in all cases throughout the research. But the mean increased in keratinized animal tissue dimension was 3 mm (range, 3–4 mm), and no vital changes in depths were ascertained. According to our findings, we can terminate that EVPOME is useful for oral application and has the flexibility to reinforce keratinized gingiva. More randomized clinical trials in this field should be performed to demonstrate other dimensions of tissue

Poor color match, donor-site morbidity, lack of

will result in microstomia and associated purposeful deficits in speech and swallowing

Long recovery, donor-site morbidity, lack of performance, poor color match/esthetics, needs

Needs long immunosuppression, facet effects of immunosuppression, long and troublesome recovery, donor accessibility, needs specialized

bulk, no performance

specialized surgical skills

surgical skills and facility

Sensible color match, functional Restricted quantity, might need staged surgeries,

**Approach Advantages Disadvantages**

accessibility

tissue, esthetics

Simply harvested, vital tissue

Wonderful tube peduncle, applicable tissue thickness, and a technique to suspend the lip with the incorporated tendon

Sensible color match, purposeful

**Table 3.** The advantages and downsides of the contemporary approach to soft tissue reconstruction [66].

The maintenance of associate degree sufficient strip of attached gingiva includes a minimum of 2 mm of keratinized gingiva and has revealed that it could be necessary for preservation of periodontal tissue [68, 69]. Historically, FGGs' associate degreed grafts which were taken from connective tissue are applied to gain sufficient strip of attached gingiva [70]. Unfortunately, clinical evidence about the use of autologous and allogenic products confronts to some problems about FGGs, i.e., problems about morbidity around donor region and also amount of tissue for graft which is restricted [71]. Nevins [72] applied treatment by bilayered cell for mucogingival region, and this research revealed that adequate keratinized tissue was gained; however, the amount was not more than it gained with FGGs. Interestingly, each research tried to prove that clinical evidence such as texture and color have better results when compared with FGGs. Nevertheless, application of products that have allogenic cells could have important effect on the wound bed; the impact of treatment could be as a completely unique "biologic dressing" to motivate encompassing cells. One of the reliable materials was a scaffold which is biodegradable with gingival autologous fibroblasts which are cultured [73]. Another research in this field used oral mucosa cells to transplant directly to the cornea [74].

engineering [67].

Free grafts (full-thickness skin grafts, split-thickness

282 Stem Cells in Clinical Practice and Tissue Engineering

Local advancement and

Free vascularized tissue

Allogeneic tissue transfer/

skin grafts, etc.)

motility flaps

face transplant

transfer

The temporomandibular joint (TMJ) may be a synovial joint that has for articulator motion relative to the os base and distributes the traditional stresses of perform (chewing and speaking) and parafunction (clenching and bruxism). It is usually noted as ginglymoarthrodial joint attributable to its slippy performance and hinging. The temporomandibular joint links the condyloid process (mandibular bone) to the temporal bone. The cartilage disc is the middle of mandibular condyle and the glenoid fossa eminence of the temporal bone and separates the joint area into inferior and superior compartments, each of that area unit crammed with synovial fluid [79].

Because of the advanced loading patterns that designed tissues can expertise within the TMJ, acquisition of complete style parameters from the native tissue is important. Significantly, TMJ disc, condyle, and condylar cartilage replacement area unit in nice demand attributable to these tissues' poor regenerative capability and high rate of involvement in TMD. In response, many studies characterizing the properties of those elements are performed [79]. Though glenoid fossa and articular eminence are the concern in TMD, they need not be absolutely characterized. Within the following section, structural characteristics of the TMJ tissue area unit are summarized. Application of tissue engineering in the treatment of temporomandibular joint defects is rising as a progressive choice to substitute and remove the pathological defects automatically in the near future. Historically, basics of regenerative medicine are three necessary elements, cells, stimulator factors, and scaffolds. But new technology introduced novel methodologies and recommends how to manage TMJ disk and stimulate relevant cartilage [80]. This approach includes the scaffold-free and cell-based methodology and cells and stimulators that work together. But another approach is to construct a structure and render appropriate form of engineered tissues, permitting well-designed structure and simple handling [81]. Another mechanical feature may be appropriate for designed tissue. In the best manner the chemistry of scaffold that could be degradation with matrix synthesis. But the rate of scaffold degradation depends on the nature of scaffold and might be modified by manipulation. For induction of the mass of matrix synthesis, growth factors are added to the scaffolds. Today, all engineered tissue materials are used to regenerate condyle and TMJ disc; however, similar strategies for regeneration of mandibular fossa are not successful [82].

### **7. Conclusion**

Unfortunately, limiting factors still existed; most of them could be the differences of lab environment and human body such as unknown exact dose of BMP and applicate high dose of this material to creation of bony scaffold [83]. On the other hand, unpredictable effects of BMP and complications about application of BMP together with oncogenesis. Based on these evidences, Food and Drug Administration (FDA) restricted application of BMP to sinus alveolar process augmentation in the United States; other substitute materials in maxillofacial region should be considered moral issues even if these materials reveal high level of evidence in several experimentations [84].

**Author details**

Seied Omid Keyhan1,2,3,4, Hamidreza Fallahi<sup>5</sup>

Mohammad Hosein Amirzade-Iranaq7,8,9,10\*

\*Address all correspondence to: h.amirzade@gmail.com

Education and Research Network (USERN), Tehran, Iran

Seyed Mohammad Reza Masoumi<sup>7</sup>

Medical Science, Birjand, Iran

6 Private Practice, Tehran, Iran

Academic Press: MA, USA. 2011

Scandinavica. 2017;**75**(1):1-11

als. Boca Raton: CRC Press; 2008. p. 185-211

Sciences, Tehran, Iran

Tehran, Iran

**References**

, Alireza Jahangirnia6

, Mohammad Hossein Khosravi7,8 and

1 Department of Oral & Maxillofacial Surgery, Faculty of Dentistry, Birjand University of

2 Vice Presidential Organization of Technology of the Islamic Republic of Iran, Iran

3 Stem Cell & Regenerative Medicine Network, Shahid Beheshti University of Medical

4 Cranio Maxillofacial Research Center, Tehran Dental Branch, Islamic Azad University,

5 Oral and Maxillofacial Surgery, Jundishapur University of Medical Sciences, Ahvaz, Iran

7 Student Research Committee (SRC), Baqiyatallah University of Medical Sciences, Tehran, Iran

9 Student Research Committee, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

[1] Lanza R, Langer R, Vacanti JP. Principles of Tissue Engineering. Academic Press; Elsevier

[2] Fisher MB, Mauck RL. Tissue engineering and regenerative medicine: Recent innovations and the transition to translation. Tissue Engineering Part B: Reviews. 2013;**19**(1):1-13

[3] Salih V. Biodegradable scaffolds for tissue engineering. Cellular response to biomateri-

[4] Mihaylova Z, Mitev V, Stanimirov P, Isaeva A, Gateva N, Ishkitiev N. Use of platelet concentrates in oral and maxillofacial surgery: An overview. Acta Odontologica

8 International Otorhinolaryngology Research Association (IORA), Universal Scientific

10 Universal Network of Interdisciplinary Research in Oral and Maxillofacial Surgery (UNIROMS), Universal Scientific Education and Research Network (USERN), Tehran, Iran

,

Tissue Engineering Applications in Maxillofacial Surgery http://dx.doi.org/10.5772/intechopen.70904 285

Another problem like tissue transfer decreases the chance of high-quality and priced experimentations [85], and also Ripamonti et al. study revealed that growth factor and signaling systems in animal and human are totally different and huge variation between them is observed. Unfortunately, there are few clinical trials in the maxillofacial region, but the question is what the obstacles are? The main obstacles are how to predict regenerate cells to not become oncogenesis and produce our wanted cells, how to manage signaling factors to facilitate the procedure of regeneration, and how to create the scaffold that permits cell growth in the best way. More clinical trials are needed to remove the obstacles [86].

Tissue engineering is the field that is surrounded by other fields like histology, medical engineering, and pathology that every progress in these fields could change principles of tissue engineering. Our goals are simple which are to know how to regenerate human tissues from host cells and somehow that these regenerates have desirable function and esthetics. To reach to this goal, we have long way, but today engineers progressed biocompatible scaffolds, increase the flexibility to 3D tissue constructs, and designed complex tissue for the different facial areas. The latest progress guarantees that tissue engineering is the trustworthy choice for the treatment of maxillofacial defects. In future, the role of tissue engineering will increase and become routine in surgeries.

### **Acknowledgements**

Conducting this review will not be possible without team work and cooperation. We have to thank people for their work in helping us through this way, authors of all referenced articles which are used for construction of this review, Dr. Tannaz Tafakori, Eng. MT Amirzade-Iranaq, and Mr. MS Amirzade-Iranaq with their search and companionship.

### **Conflict of interest**

Authors declare that there is no conflict of interest that may damage the integrity and validity of this research.
