**Tissue Engineering in Low Urinary Tract Reconstruction**

Chao Feng and Yue-min Xu

*Department of Urology, Shanghai Jiaotong University-Affiliated 6th People's Hospital, Shanghai, China* 

#### **1. Introduction**

424 Tissue Regeneration – From Basic Biology to Clinical Application

Yoon, B. S.&Lyons, K. M. (2004). "Multiple functions of BMPs in chondrogenesis." *J Cell* 

Yoon, B. S., Pogue, R., Ovchinnikov, D. A. (2006). "BMPs regulate multiple aspects of

Zarnett, R.&Salter, R. B. (1989). "Periosteal neochondrogenesis for biologically resurfacing

Zhao, F., Grayson, W. L., Ma, T. (2006). "Effects of hydroxyapatite in 3-D chitosan-gelatin

mineralization." *J Cell Biochem* 57(2): 218-37.

joints: its cellular origin." *Can J Surg* 32(3): 171-4.

*Biochem* 93(1): 93-103.

*Development* 133(23): 4667-78.

*Biomaterials* 27(9): 1859-67.

chondrocytes: evidence for cellular processing of Ca2+ and Pi prior to matrix

growth-plate chondrogenesis through opposing actions on FGF pathways."

polymer network on human mesenchymal stem cell construct development."

Acquired and congenital abnormalities of the lower urinary tract often require eventual reconstruction. Traditionally, different types of autologous tissue can be chosen for surgery, depending on which organ requires reconstruction. Bladder reconstruction, for example, is usually performed with intestinal tissue while urethral reconstruction can us buccal mucosa, lingual mucosa, colonic mucosa or prepuce skin. However, the problems of a shortage of patients' own tissues, and of nmany complications related to surgery, have not yet been resolved. There is therefore an effort to obtain sufficient tissue resources, to involve fewer complications, to reduce surgery to relatively minor invasion and to achieve better surgical outcomes. These goals may be attainable by the use of tissue engineering techniques.

Over the last 50 years, tissue engineering techniques for low urinary tract regeneration have been applied successfully in a variety of animal models and clinical patients. Rapid advancement has been made in this field, which has broadened the theoretical options for the future of low urinary tract reconstruction. These developments include improvements in cell culture techniques, such as the development of cell resources and identification of markers to isolate and characterize specific cell types. Many new types of natural and synthetic biomaterials for use as scaffold components have been created (1). In addition to these, the applications of nanotechnology and bioreactors have been strengthened within recent decades. Here, we review the literature on the basic principles and latest developments of tissue engineering technologies in lower urinary tract reconstruction.

#### **2. Basic knowledge of tissue engineering in low urinary tract**

#### **2.1 Cell sources**

#### **2.1.1 Autologous stromal cells**

Because epithelial cells are one of the most important components of the lower urinary tract, optimizing sources for them have always been a popular focus of investigators. Traditionally, urothelial cells obtained from bladder or urethra have often been used in previous studies (Fig 1a) (2,3). Although this technique exploits homotypy between the graft cells and host, it involves injury to the genitourinary tract and the operation is complicated.

Tissue Engineering in Low Urinary Tract Reconstruction 427

problem during culturing of these, we advise that a velocity sedimentation method be used to evaluate the purification of smooth muscle cells. Of course, obvious trauma after the procedure is also the shortcoming of this method. As a result of these problems, smooth

Stem cells from bone marrow (BMSC) have been characterized as being either hematopoietic or mesenchymal. They are easily isolated due to their affinity with and adherence to plastic dishes. Their ability to proliferate ensures that even a small number of BMSC multiply into millions of cells under the right culture conditions. Another merit of these cells is that they do not express MHC II, rendering them nonimmunogenic and thereby eliminating possible graft rejection (8). Previous studies showed that BMSCs contained higher concentrations of -SM actin than did bladder SMC. Meanwhile, BMSCs showed strong response to the Ca2+ ionophore, whereas fibroblasts did not contract their baseline even in the presence of calcium. Those results indicate that BMSCs and smooth muscle cells from low urinary tract are very similar (9). Therefore, BMSCs may serve as an alternative cell source in lower

Stem cells from adipose tissue (ADSCs) have also been popular in tissue engineering research. Adipose tissue is derived from embryonic mesodermal precursors and it contains multipotent progenitor cells that are capable of differentiating into mesenchymal tissue. Since adipose tissue contains 100-1,000 times more pluripotent cells per cubic centimeter

Fig. 3. Myogenic differentiation of human BMSCs using SMC-derived CM. Human BMSCs (p4) were stained with a-SMA (a, e, i), calponin (b, f, j), desmin (c, g, k), and myosin (d, h, l) antibodies without induction as negative control (a–d) and with induction for 14 days (e–h). SMCs were also stained with the same antibodies as a positive control

muscle cells are being replaced by muscle stem cells in urinary reconstruction.

**2.1.2 Bone marrow and adipose derived stem cells** 

urinary tract tissue engineering.

(i–l). (Picture from ref 11)

Fu, *et al.* chose epidermal cells as graft cells because of its abundant resources; they can be obtained by a less invasive method than the traditional method of bladder or urethral biopsy followed by dissection of transitional cells. The results suggest that the epidermal cells can transform to transitional epithelial cells under the influence of the urethral or bladder environment (4). From our experience, we suggest using the oral keratinocytes, such as buccal keratinocytes and lingual keratinocytes, as a source of epithelial cells, (Fig 1b,c). Such cells express the -defensin, IL-8, which can mediate an innate immune response against microbes (5). Therefore, compound grafts were easily resisted infection both in vitro and in vivo. In addition, these oral keratinocytes expressed AE1/AE3, which is similar to epidermal cells or urothelial cells in a previous report (6). However, 3T3 cells are usually needed as a feeder layer when culturing oral keratinocytes. The purification of oral keratinocytes therefore needs to be should be improved before clinical application.

Fig. 1. Morphology the different kinds of epithelial seeding cells. a. Bladder urethelial cells; b. buccal keratinocytes; c. lingual keratinocytes

To construct 3D bladder or urethral tissue, smooth muscle cell is also necessary. Previously, bladder smooth muscle cells were used for tissue engineering bladder reconstruction (Fig2a). The corpora cavernosa smooth muscle cells were used for constructing the corpora spongiosum, which is one of the most important components of the penile urethra (Fig2b). The advantage of using those cells is that some angiogenic growth factors and their receptors, such as Flk-1 and VEGF, are present in smooth muscle cells. They might contribute to the angiogenesis of bladder or urethral tissue (7). Since contamination by fibroblast cells is a

Fig. 2. Morphology the different kinds of smooth muscle cells. a. Bladder smooth muscle cells; b. corpora cavernosa smooth muscle cells

Fu, *et al.* chose epidermal cells as graft cells because of its abundant resources; they can be obtained by a less invasive method than the traditional method of bladder or urethral biopsy followed by dissection of transitional cells. The results suggest that the epidermal cells can transform to transitional epithelial cells under the influence of the urethral or bladder environment (4). From our experience, we suggest using the oral keratinocytes, such as buccal keratinocytes and lingual keratinocytes, as a source of epithelial cells, (Fig 1b,c). Such cells express the -defensin, IL-8, which can mediate an innate immune response against microbes (5). Therefore, compound grafts were easily resisted infection both in vitro and in vivo. In addition, these oral keratinocytes expressed AE1/AE3, which is similar to epidermal cells or urothelial cells in a previous report (6). However, 3T3 cells are usually needed as a feeder layer when culturing oral keratinocytes. The purification of oral

keratinocytes therefore needs to be should be improved before clinical application.

Fig. 1. Morphology the different kinds of epithelial seeding cells.

Fig. 2. Morphology the different kinds of smooth muscle cells.

a. Bladder smooth muscle cells; b. corpora cavernosa smooth muscle cells

a. Bladder urethelial cells; b. buccal keratinocytes; c. lingual keratinocytes

To construct 3D bladder or urethral tissue, smooth muscle cell is also necessary. Previously, bladder smooth muscle cells were used for tissue engineering bladder reconstruction (Fig2a). The corpora cavernosa smooth muscle cells were used for constructing the corpora spongiosum, which is one of the most important components of the penile urethra (Fig2b). The advantage of using those cells is that some angiogenic growth factors and their receptors, such as Flk-1 and VEGF, are present in smooth muscle cells. They might contribute to the angiogenesis of bladder or urethral tissue (7). Since contamination by fibroblast cells is a problem during culturing of these, we advise that a velocity sedimentation method be used to evaluate the purification of smooth muscle cells. Of course, obvious trauma after the procedure is also the shortcoming of this method. As a result of these problems, smooth muscle cells are being replaced by muscle stem cells in urinary reconstruction.

#### **2.1.2 Bone marrow and adipose derived stem cells**

Stem cells from bone marrow (BMSC) have been characterized as being either hematopoietic or mesenchymal. They are easily isolated due to their affinity with and adherence to plastic dishes. Their ability to proliferate ensures that even a small number of BMSC multiply into millions of cells under the right culture conditions. Another merit of these cells is that they do not express MHC II, rendering them nonimmunogenic and thereby eliminating possible graft rejection (8). Previous studies showed that BMSCs contained higher concentrations of -SM actin than did bladder SMC. Meanwhile, BMSCs showed strong response to the Ca2+ ionophore, whereas fibroblasts did not contract their baseline even in the presence of calcium. Those results indicate that BMSCs and smooth muscle cells from low urinary tract are very similar (9). Therefore, BMSCs may serve as an alternative cell source in lower urinary tract tissue engineering.

Stem cells from adipose tissue (ADSCs) have also been popular in tissue engineering research. Adipose tissue is derived from embryonic mesodermal precursors and it contains multipotent progenitor cells that are capable of differentiating into mesenchymal tissue. Since adipose tissue contains 100-1,000 times more pluripotent cells per cubic centimeter

Fig. 3. Myogenic differentiation of human BMSCs using SMC-derived CM. Human BMSCs (p4) were stained with a-SMA (a, e, i), calponin (b, f, j), desmin (c, g, k), and myosin (d, h, l) antibodies without induction as negative control (a–d) and with induction for 14 days (e–h). SMCs were also stained with the same antibodies as a positive control (i–l). (Picture from ref 11)

Tissue Engineering in Low Urinary Tract Reconstruction 429

Fig. 4. Morphology of urine-derived stem cells obtained from upper urinary tract

addition of uro-epithelial medium. Scale bar shown is 100 mm (Picture from ref 14).

**2.2 Biomaterials** 

and degradation rates (15).

to the original organs.

**2.2.1 Traditional biomaterials** 

(USC-UUT) with differentiation. a. non-treated USC; b. The shape of -UUT changed from an oval to a spindle shape with the addition of myogenic medium; c. a cuboidal shape with the

Creating an ideal biomaterial for lower urinary tract reconstruction has been an aspiration of urologists for over a century. An excellent biomaterial for tissue engineering should possess optimal mechanical properties, good biocompatibility, suitable three dimensional structures

Traditionally, biomaterials can be classified into naturally derived materials, including chitosan, collagen; acellular matrix , such as small intestine submucosa (SIS), bladder acellular matrix (BAMG), acellular corpous spongious matrix (ACSM) and urethral extra matrix (UEM) , as well as synthetic materials, such as PGA and PLGA. Most of them have been used in animal models and human subjects, which will be discussed in the later section. Brehmer provides a useful classification of scaffolds into carrier-, fleece- and sponge-types, according to the structure of biomaterials (16) Carrier-type scaffolds are fiber meshes with very small pore sizes (<15 m). The pore size of the sponge-type scaffolds is greater than 15 m. Fleece-type scaffolds have huge interfilamentary spaces (200 m). In our previous study, we compared the dimensional structures of SIS, BAMG, handmade PGA mesh and ACSM. SEM demonstrated that the pore size of the PGA (>200 m) was the largest among all biomaterials. The surface pore sizes in SIS were significantly larger than BAMG (58.32 ± 10.31 m vs 6.77 ± 0.49 m; P < 0.05). Although a looser structure of BAMG could be seen with H&E staining, its pore sizes in surface views were smaller than those of ACSM (6.77 ± 0.49 m vs 11.12 ± 1.43 m; P < 0.05). An obvious difference of pore diameters in ACSM could be distinguished between urethral surface and cavernosal surface. (2.04 ± 0.32 m vs 11.12± 1.43 m; P< 0.05)(Fig 5)(17). This data can guide the following cell seeding procedure, since cellular growth and infiltration are strongly related to the scaffold's pore sizes. Of course, it should be noted that the structure of PGA or PLGA can be controlled now with the development of electrospin techniques. Therefore, the dimensional structure of synthetic materials is becoming more similar to the naturally derived scaffolds, and even

than does bone marrow, it is easier to obtain ADCSs than other kinds of adult stem cells. Of course, immunoprivilege is also the advantage of this kind of cell. For these reasons, many investigators have selected ADSCs as an ideal source of seeding cells in lower urinary reconstruction, such as repair of bladder and urethra (10).

In many studies, mesenchymal cells have been found to differentiate into many different lineages, such as chondrocytes, osteoblasts, adipocytes, neurons and myoblasts. To urologists, the most interesting thing is the possibility for differentiation of BMSCs or ADSCs into smooth muscle cells and keratinocytes. According to previous reports, these stem cells can acquire a smooth muscle cell phenotype, staining positively for -SMA, myosin and calponin after being cultured in conditioned medium. Also, culturing in the presence of other myogenic growth factors, such as PDFF-BB,HGF,TGF-, can also lead to a phenotypic profile of smooth muscle cells (Fig 3) (8,11). Another group has demonstrated the differentiation of marked BMSC into urothelial cells on a seeded scaffold in porcine bladder augmentation, suggesting that mesenchymal stem cells can be made into urothelial cells. However, few additional reports support this result. Since the BMSCs and ADSCs are derived from the mesodermal lineage, more evidence is needed to support that ectodermal lineage cells can be induced from mesenchymal stem cells, such as BMSCs and ADSCs.

#### **2.1.3 Other seeding cells**

As well as the autologous stromal cells, BMSCs and ADSCs, other kinds of seeding cells have also shown possibilities for lower urinary tract reconstruction. Drewa (12) *et al.* used hair follicle stem cells for bladder regeneration in rats. This type of cell is CD34 positive, which facilitates the isolation of live epithelial cells with stem cell characterisitcs. In their study, Drewa *et al.* concluded that pluripotent stem cells within rodent hair follicle can differentiate into neurons, glia, keratinocytes and smooth muscle cells. They used an acellular matrix seeded with those cells and achieved a successful bladder wall reconstruction. Further research should be focused on better characterization of these cell populations and on the exact mechanism by which these cells enhance bladder regeneration.

Zhang's study focused on a subpopulation of cells isolated from naturally voided urine (13). This kind of cell demonstrated features typical of progenitor/stem cells, including expression of MSC and pericyte cell surface markers and clonogenic, multipotential, and plastic adhensive capacity. Furthermore, recent study showed that these cells have the capability to differentiate into the urothelial and smooth muscle cells (Fig 4)(14). The latest study has demonstrated the feasibility of forming a tissue-engineered conduit for use in urinary diversion by generating scaffolds seeded with human urine-derived stem cells.

Other cells, such as human amniotic fluid stem cells (AFS), human embryonic stem cells (ES) and human induced pluripotent stem cells (iPS), have also shown a potential for application in lower urinary reconstruction. However, most reports have been rather preliminary investigations. Several key points still need to be studied in depth before the cells can be used in patients.

Fig. 4. Morphology of urine-derived stem cells obtained from upper urinary tract (USC-UUT) with differentiation. a. non-treated USC; b. The shape of -UUT changed from an oval to a spindle shape with the addition of myogenic medium; c. a cuboidal shape with the addition of uro-epithelial medium. Scale bar shown is 100 mm (Picture from ref 14).

#### **2.2 Biomaterials**

428 Tissue Regeneration – From Basic Biology to Clinical Application

than does bone marrow, it is easier to obtain ADCSs than other kinds of adult stem cells. Of course, immunoprivilege is also the advantage of this kind of cell. For these reasons, many investigators have selected ADSCs as an ideal source of seeding cells in lower urinary

In many studies, mesenchymal cells have been found to differentiate into many different lineages, such as chondrocytes, osteoblasts, adipocytes, neurons and myoblasts. To urologists, the most interesting thing is the possibility for differentiation of BMSCs or ADSCs into smooth muscle cells and keratinocytes. According to previous reports, these stem cells can acquire a smooth muscle cell phenotype, staining positively for -SMA, myosin and calponin after being cultured in conditioned medium. Also, culturing in the presence of other myogenic growth factors, such as PDFF-BB,HGF,TGF-, can also lead to a phenotypic profile of smooth muscle cells (Fig 3) (8,11). Another group has demonstrated the differentiation of marked BMSC into urothelial cells on a seeded scaffold in porcine bladder augmentation, suggesting that mesenchymal stem cells can be made into urothelial cells. However, few additional reports support this result. Since the BMSCs and ADSCs are derived from the mesodermal lineage, more evidence is needed to support that ectodermal lineage cells can be induced from mesenchymal stem cells, such as BMSCs and ADSCs.

As well as the autologous stromal cells, BMSCs and ADSCs, other kinds of seeding cells have also shown possibilities for lower urinary tract reconstruction. Drewa (12) *et al.* used hair follicle stem cells for bladder regeneration in rats. This type of cell is CD34 positive, which facilitates the isolation of live epithelial cells with stem cell characterisitcs. In their study, Drewa *et al.* concluded that pluripotent stem cells within rodent hair follicle can differentiate into neurons, glia, keratinocytes and smooth muscle cells. They used an acellular matrix seeded with those cells and achieved a successful bladder wall reconstruction. Further research should be focused on better characterization of these cell populations and on the exact mechanism by which these cells enhance bladder

Zhang's study focused on a subpopulation of cells isolated from naturally voided urine (13). This kind of cell demonstrated features typical of progenitor/stem cells, including expression of MSC and pericyte cell surface markers and clonogenic, multipotential, and plastic adhensive capacity. Furthermore, recent study showed that these cells have the capability to differentiate into the urothelial and smooth muscle cells (Fig 4)(14). The latest study has demonstrated the feasibility of forming a tissue-engineered conduit for use in urinary diversion by generating scaffolds seeded with human urine-derived stem

Other cells, such as human amniotic fluid stem cells (AFS), human embryonic stem cells (ES) and human induced pluripotent stem cells (iPS), have also shown a potential for application in lower urinary reconstruction. However, most reports have been rather preliminary investigations. Several key points still need to be studied in depth before the cells can be

reconstruction, such as repair of bladder and urethra (10).

**2.1.3 Other seeding cells** 

regeneration.

cells.

used in patients.

Creating an ideal biomaterial for lower urinary tract reconstruction has been an aspiration of urologists for over a century. An excellent biomaterial for tissue engineering should possess optimal mechanical properties, good biocompatibility, suitable three dimensional structures and degradation rates (15).

#### **2.2.1 Traditional biomaterials**

Traditionally, biomaterials can be classified into naturally derived materials, including chitosan, collagen; acellular matrix , such as small intestine submucosa (SIS), bladder acellular matrix (BAMG), acellular corpous spongious matrix (ACSM) and urethral extra matrix (UEM) , as well as synthetic materials, such as PGA and PLGA. Most of them have been used in animal models and human subjects, which will be discussed in the later section. Brehmer provides a useful classification of scaffolds into carrier-, fleece- and sponge-types, according to the structure of biomaterials (16) Carrier-type scaffolds are fiber meshes with very small pore sizes (<15 m). The pore size of the sponge-type scaffolds is greater than 15 m. Fleece-type scaffolds have huge interfilamentary spaces (200 m). In our previous study, we compared the dimensional structures of SIS, BAMG, handmade PGA mesh and ACSM. SEM demonstrated that the pore size of the PGA (>200 m) was the largest among all biomaterials. The surface pore sizes in SIS were significantly larger than BAMG (58.32 ± 10.31 m vs 6.77 ± 0.49 m; P < 0.05). Although a looser structure of BAMG could be seen with H&E staining, its pore sizes in surface views were smaller than those of ACSM (6.77 ± 0.49 m vs 11.12 ± 1.43 m; P < 0.05). An obvious difference of pore diameters in ACSM could be distinguished between urethral surface and cavernosal surface. (2.04 ± 0.32 m vs 11.12± 1.43 m; P< 0.05)(Fig 5)(17). This data can guide the following cell seeding procedure, since cellular growth and infiltration are strongly related to the scaffold's pore sizes. Of course, it should be noted that the structure of PGA or PLGA can be controlled now with the development of electrospin techniques. Therefore, the dimensional structure of synthetic materials is becoming more similar to the naturally derived scaffolds, and even to the original organs.

Tissue Engineering in Low Urinary Tract Reconstruction 431

The mechanical properties of biomaterials are also key to successful reconstruction of the lower urinary tract. For urethral reconstruction, a uniaxial mechanical test is necessary to evaluate the scaffold. In our previous study, all biomaterials exhibited the classic biological nonlinear stress–strain response (Fig. 7) in a mechanical test. The ACSM showed good response in Young's modulus and breaking stress, these being better than in other scaffolds, even the normal rabbit urethra. For the bladder, physiological loading of the tissue involves compressive loads perpendicular to the bladder surface, induced by urine and surrounding pelvic tissues, so biaxial mechanical testing is more realistic. In addition, a burst experiment

Fig. 7. Stress–Strain curves of various biomaterials. (A) Normal rabbit urethra; (B) SIS;

Fig. 8. Picture of ball-burst test with a ruptured test material. Arrow points at the rupture

Since inherit weaknesses always exist in traditional biomaterials, many modified

should also be considered (18) (Fig 8)

(C) 4-layer SIS; (D) BAMG; (E) ACSM; (F) PGA

**2.2.2 Modified & advanced biomaterials** 

biomaterials have been studied to avoid them.

site from (ref 18)

Fig. 5. EMS examination of different materials' surface. (A) urethral surface of ACSM, EMS×5,000, (B) cavernosal surface of ACSM,EMS×5,000 (C) surface of BAMG, EMS×5,000. (D) surface of SIS, EMS\_200. (E) surface of PGA, EMS×200.

Fig. 6. Metabolic activity of CCSMCs cultured with extracts of various biomaterials or cultured directly in normal medium at 1, 3, 8, and 10 days, as determined by MTT assays. The difference between biomaterials and negative controls was not statistically significant

To address the issue of biocompatibility, our study used the MTT assay technique to evaluate cytotoxicity of different kinds of biomaterials. There were no statistically significant differences in MTT results between the cells cultured with biomaterial extracts and with controls (Fig 6). Thus, we may suggest that all scaffolds could be used safely for lower urinary tract reconstruction.

Fig. 5. EMS examination of different materials' surface. (A) urethral surface of ACSM, EMS×5,000, (B) cavernosal surface of ACSM,EMS×5,000 (C) surface of BAMG, EMS×5,000. (D) surface of SIS, EMS\_200. (E) surface of PGA, EMS×200.

Fig. 6. Metabolic activity of CCSMCs cultured with extracts of various biomaterials or cultured directly in normal medium at 1, 3, 8, and 10 days, as determined by MTT assays. The difference between biomaterials and negative controls was not statistically significant

urinary tract reconstruction.

To address the issue of biocompatibility, our study used the MTT assay technique to evaluate cytotoxicity of different kinds of biomaterials. There were no statistically significant differences in MTT results between the cells cultured with biomaterial extracts and with controls (Fig 6). Thus, we may suggest that all scaffolds could be used safely for lower The mechanical properties of biomaterials are also key to successful reconstruction of the lower urinary tract. For urethral reconstruction, a uniaxial mechanical test is necessary to evaluate the scaffold. In our previous study, all biomaterials exhibited the classic biological nonlinear stress–strain response (Fig. 7) in a mechanical test. The ACSM showed good response in Young's modulus and breaking stress, these being better than in other scaffolds, even the normal rabbit urethra. For the bladder, physiological loading of the tissue involves compressive loads perpendicular to the bladder surface, induced by urine and surrounding pelvic tissues, so biaxial mechanical testing is more realistic. In addition, a burst experiment should also be considered (18) (Fig 8)

Fig. 7. Stress–Strain curves of various biomaterials. (A) Normal rabbit urethra; (B) SIS; (C) 4-layer SIS; (D) BAMG; (E) ACSM; (F) PGA

Fig. 8. Picture of ball-burst test with a ruptured test material. Arrow points at the rupture site from (ref 18)

#### **2.2.2 Modified & advanced biomaterials**

Since inherit weaknesses always exist in traditional biomaterials, many modified biomaterials have been studied to avoid them.

Tissue Engineering in Low Urinary Tract Reconstruction 433

Fig. 9. Harvesting of the matured construct 3 wk after implantation in the omentum (ref 21)

Fig. 10. a. Disassembled urinary bladder bioreactor. I. This chamber will be subjected to controlled pressure and hence would mimic in vitro the urinary bladder chamber. II. Tissue

engineered construct ring. III. Compliance chamber (cell culture medium will be recirculated to accommodate the expansion of the scaffold upon pressure generation).

b Interlocking discs for cell-seeded scaffold. c Assembled bioreactor (ref 23)

To enhance angiogenesis, some investigators have modified traditional matrices by incorporating heparin and subsequently loading the heparinized matrices with VEGF. Preliminary studies have shown that this loading of the matrices with VEGF increases the induction of microvessels in both heparinized and non-heparinized matrices, the effect being largest in the case of the heparinized matrices (19).

In order to control the three dimensional structure and degradation rate of synthetic scaffolds such as PGA or PLGA, electrospin techniques are often considered for tissue engineering in lower urinary tract reconstruction. Various materials have been examined for their ability to support cellular adhension, proliferation and formation of a multilayerd urothelium. The results provide the evidence that electrospinning scaffolds show significant benefitsa over commonly used acellular materials in vitro, and suggest that they should be further examined in vivo (20).

#### **2.3 Advanced technique**

#### **2.3.1 Bioreactor**

The bioreactor is a device that provides a fluid environment for the growth of cells for various applications, such as industrial fermentation and cell culturing. Bioreactors should be introduced in tissue engineering to optimize, through fluid shear, oxygenation and the supply of nutrients, the growth of cells on a 3D scaffold. This approach has been shown to result in better tissue-like constructs than do conventional static culture conditions (21). It is possible to use in vivo graft sites as 'bioreactors' that feature flowing fluids (blood). An example that is commonly used in tissue engineering for lower urinary reconstruction is the greater omentum. Baumert et al (21) used urothelial and smooth muscle cells to seed a sphere-shaped small intestinal submucosa matrix, which was transferred into the omentum after 3wk of cell growth. By this approach, they obtained tissue engineered bladder with a wall thickness was 4 mm. The construct presented a multilayer urothelium on the lumial aspect and deeper fascicles of organized tissue composed of differentiated smooth muscle cells and mature fibroblasts. There was no evidence of inflammation or necrosis (Fig 9). Gu et al (22) implanted 8Fr silastic tubes into the peritoneal cavity of a rabbit. Those tubes were harvested and the tubular tissue covering the tubes was reverted. A pendulous urethral segment of 1.5 cm long was totally excised and urethroplasty was performed with the reverted tubular tissue in an end-to-end fashion. Finally, the results of study showed that the recipients' peritoneal cavity can be used as bioreactor for tissue engineering urethral reconstruction.

More manufactured bioreactors have been designed for tissue engineering bladder. In order to mimic the dynamics of the urinary bladder, bioreactors that imitate the filling and emptying of a normal bladder have been suggested. A bladder bioreactor built this way should be able to recapitulate those dynamics while providing a cellular environment that facilitates cell-cell and cell-matrix interactions. Under the mechanical stimulation from bioreactor, the physiological and mechanical properties of the bladder can be improved. The growth behavior of urothelial cells and bladder smooth cells can be changed, resulting in the cells undergoing adaptive changes in mechanically-stimulated environment (23, 24).

To enhance angiogenesis, some investigators have modified traditional matrices by incorporating heparin and subsequently loading the heparinized matrices with VEGF. Preliminary studies have shown that this loading of the matrices with VEGF increases the induction of microvessels in both heparinized and non-heparinized matrices, the effect

In order to control the three dimensional structure and degradation rate of synthetic scaffolds such as PGA or PLGA, electrospin techniques are often considered for tissue engineering in lower urinary tract reconstruction. Various materials have been examined for their ability to support cellular adhension, proliferation and formation of a multilayerd urothelium. The results provide the evidence that electrospinning scaffolds show significant benefitsa over commonly used acellular materials in vitro, and suggest that they should be

The bioreactor is a device that provides a fluid environment for the growth of cells for various applications, such as industrial fermentation and cell culturing. Bioreactors should be introduced in tissue engineering to optimize, through fluid shear, oxygenation and the supply of nutrients, the growth of cells on a 3D scaffold. This approach has been shown to result in better tissue-like constructs than do conventional static culture conditions (21). It is possible to use in vivo graft sites as 'bioreactors' that feature flowing fluids (blood). An example that is commonly used in tissue engineering for lower urinary reconstruction is the greater omentum. Baumert et al (21) used urothelial and smooth muscle cells to seed a sphere-shaped small intestinal submucosa matrix, which was transferred into the omentum after 3wk of cell growth. By this approach, they obtained tissue engineered bladder with a wall thickness was 4 mm. The construct presented a multilayer urothelium on the lumial aspect and deeper fascicles of organized tissue composed of differentiated smooth muscle cells and mature fibroblasts. There was no evidence of inflammation or necrosis (Fig 9). Gu et al (22) implanted 8Fr silastic tubes into the peritoneal cavity of a rabbit. Those tubes were harvested and the tubular tissue covering the tubes was reverted. A pendulous urethral segment of 1.5 cm long was totally excised and urethroplasty was performed with the reverted tubular tissue in an end-to-end fashion. Finally, the results of study showed that the recipients' peritoneal cavity can be used as bioreactor for tissue engineering urethral

More manufactured bioreactors have been designed for tissue engineering bladder. In order to mimic the dynamics of the urinary bladder, bioreactors that imitate the filling and emptying of a normal bladder have been suggested. A bladder bioreactor built this way should be able to recapitulate those dynamics while providing a cellular environment that facilitates cell-cell and cell-matrix interactions. Under the mechanical stimulation from bioreactor, the physiological and mechanical properties of the bladder can be improved. The growth behavior of urothelial cells and bladder smooth cells can be changed, resulting in the cells undergoing adaptive changes in mechanically-stimulated

being largest in the case of the heparinized matrices (19).

further examined in vivo (20).

**2.3 Advanced technique** 

**2.3.1 Bioreactor** 

reconstruction.

environment (23, 24).

Fig. 9. Harvesting of the matured construct 3 wk after implantation in the omentum (ref 21)

Fig. 10. a. Disassembled urinary bladder bioreactor. I. This chamber will be subjected to controlled pressure and hence would mimic in vitro the urinary bladder chamber. II. Tissue engineered construct ring. III. Compliance chamber (cell culture medium will be recirculated to accommodate the expansion of the scaffold upon pressure generation). b Interlocking discs for cell-seeded scaffold. c Assembled bioreactor (ref 23)

Tissue Engineering in Low Urinary Tract Reconstruction 435

As well as the electrospinning technique mentioned above, another useful nanotechnique is the use of nanoparticles as a delivery system. Mondalek et al (27) investigated the use of PLGA nanoparticles to alter the permeability of SIS scaffolds. Preliminary results indicated that particles ranging from 200 to 500 nm would become imbedded in the SIS scaffold. Particles below this size range would pass through the graft and not become entrapped, and particles above this size range could not penetrate the scaffold. Those results provided the possibility of using nanoparticles to deliver growth factors into seeded cells and scaffolds, to

Table 1. Nanotechnology Approaches to Increase Bladder Cell Functions (ref 26)

**3. Applications of tissue engineering in the lower urinary tract** 

The limitation of oxygen diffusion has led to the general concept that cell or tissue components may not be implanted in large volumes. Many efforts have been made to overcome this limitation. Recently, implantable oxygen releasing biomaterials have been developed in order to provide a sustained release of oxygen to cells and tissues with the goal of prolonging tissue survival and decreasing necrosis (28). In those studies, an oxygen rich compound of sodium percarbonate or calcium peroxide was incorporated into films or 3D constructs of PLGA and used for in situ production of oxygen. In vitro, release of oxygen could be observed from the film more than 24h. Furthermore, these biomaterials were able to extend cell viability growth under hypoxic conditions. Those findings indicate that the use of oxygen generating biomaterials may enhance the scaffold neovascularization after implantation (29). All results suggested that oxygen generating scaffolds can be used for

Congenital disorders, cancer or trauma can lead to obvious bladder damage. For patients with these problems, bladder reconstructive procedures may be considered. Although gastrointestinal segments are commonly used for bladder augmentation or replacement, multiple complications cannot yet be completely avoided; they include infection, metabolic disturbance and ureolithiasis. A number of animal studies and even clinical experiences have, however, shown the possibility of using tissue engineering techniques to reconstruct bladder tissue. In the laboratory, tissue could be engineered to have function equivalent to

enhance the regeneration of lower urinary tract.

**2.3.3 Oxygen generating scaffolds** 

lower urinary tract reconstruction in the future.

**3.1 Bladder reconstruction** 

Fig. 11. The bioreactor system. A: A diagram of the disassembled bioreactor, showing the 2 pressure chambers and 2 culture chambers, separated by 3 interlocking rings with elastic membrane. Every interlocking ring was 2 interlocking disks that hold the cell-seeded membrane, which was glued by -cyanoacrylate. B: The assembled bioreactor with the ports, to which tubing would be attached for medium flow and pressure monitoring. The red parts are culture chambers, blue parts are pressure chambers. C: The assembled culture chambers, the dashed frame showing the pressure P1 and P2 on both sides of cell-seeded membrane. the elastic membrane deformation was driven by pressure difference (P1-P2). (ref 24)

#### **2.3.2 Nanotechnology**

Nanotechnology has largely emerged in the last decade of the 20th century as a potential new enabling technology for medicine. For bladder reconstruction, this technology provides a new set of tools to solve many problems that may encountered during the reconstructive procedure. Especially, the incorporation of nanotechnology into bladder tissue engineering materials provides for better bladder materials. Recent published work has demonstrated that increasing of material surface roughness at the nanoscale can improve the adsorption of select proteins important for bladder cell functions (25). Furthermore, some reports showed more bladder smooth muscle cell attachment and growth on polystyrene nanofiber scaffolds fabricated, using an electrospinning technique, to possess surface features at the nanoscale. Cellular adhesive and proliferative ability of keratinocytes were also improved in the nanoscaled scaffold (26).(Table1)

Fig. 11. The bioreactor system. A: A diagram of the disassembled bioreactor, showing the 2 pressure chambers and 2 culture chambers, separated by 3 interlocking rings with elastic membrane. Every interlocking ring was 2 interlocking disks that hold the cell-seeded membrane, which was glued by -cyanoacrylate. B: The assembled bioreactor with the ports, to which tubing would be attached for medium flow and pressure monitoring. The red parts are culture chambers, blue parts are pressure chambers. C: The assembled culture chambers, the dashed frame showing the pressure P1 and P2 on both sides of cell-seeded membrane. the elastic membrane deformation was driven by pressure difference (P1-P2).

Nanotechnology has largely emerged in the last decade of the 20th century as a potential new enabling technology for medicine. For bladder reconstruction, this technology provides a new set of tools to solve many problems that may encountered during the reconstructive procedure. Especially, the incorporation of nanotechnology into bladder tissue engineering materials provides for better bladder materials. Recent published work has demonstrated that increasing of material surface roughness at the nanoscale can improve the adsorption of select proteins important for bladder cell functions (25). Furthermore, some reports showed more bladder smooth muscle cell attachment and growth on polystyrene nanofiber scaffolds fabricated, using an electrospinning technique, to possess surface features at the nanoscale. Cellular adhesive and proliferative ability of keratinocytes were also improved in the

(ref 24)

**2.3.2 Nanotechnology** 

nanoscaled scaffold (26).(Table1)

As well as the electrospinning technique mentioned above, another useful nanotechnique is the use of nanoparticles as a delivery system. Mondalek et al (27) investigated the use of PLGA nanoparticles to alter the permeability of SIS scaffolds. Preliminary results indicated that particles ranging from 200 to 500 nm would become imbedded in the SIS scaffold. Particles below this size range would pass through the graft and not become entrapped, and particles above this size range could not penetrate the scaffold. Those results provided the possibility of using nanoparticles to deliver growth factors into seeded cells and scaffolds, to enhance the regeneration of lower urinary tract.


Table 1. Nanotechnology Approaches to Increase Bladder Cell Functions (ref 26)

#### **2.3.3 Oxygen generating scaffolds**

The limitation of oxygen diffusion has led to the general concept that cell or tissue components may not be implanted in large volumes. Many efforts have been made to overcome this limitation. Recently, implantable oxygen releasing biomaterials have been developed in order to provide a sustained release of oxygen to cells and tissues with the goal of prolonging tissue survival and decreasing necrosis (28). In those studies, an oxygen rich compound of sodium percarbonate or calcium peroxide was incorporated into films or 3D constructs of PLGA and used for in situ production of oxygen. In vitro, release of oxygen could be observed from the film more than 24h. Furthermore, these biomaterials were able to extend cell viability growth under hypoxic conditions. Those findings indicate that the use of oxygen generating biomaterials may enhance the scaffold neovascularization after implantation (29). All results suggested that oxygen generating scaffolds can be used for lower urinary tract reconstruction in the future.

#### **3. Applications of tissue engineering in the lower urinary tract**

#### **3.1 Bladder reconstruction**

Congenital disorders, cancer or trauma can lead to obvious bladder damage. For patients with these problems, bladder reconstructive procedures may be considered. Although gastrointestinal segments are commonly used for bladder augmentation or replacement, multiple complications cannot yet be completely avoided; they include infection, metabolic disturbance and ureolithiasis. A number of animal studies and even clinical experiences have, however, shown the possibility of using tissue engineering techniques to reconstruct bladder tissue. In the laboratory, tissue could be engineered to have function equivalent to

Tissue Engineering in Low Urinary Tract Reconstruction 437

Fig. 13. Gross appearance of cell seeded constructs at 7 and 28 days post seeding. (A)

Collagen Gels, 7 days. (B) BAM, 7days. (C) Collagen Gels, 28 days. (D) BAM, 28 days. Group A: SMCs only. Group C: same side co-culture. Group D: opposite side co-culture (ref 20)

to treat the acelluar matrix for bladder tissue engineering reconstruction in vitro (35). This method led to high porosity on the surface of the matrix with about 75% of normal strength. After 3-D dynamic culture, cells could penetrate deeper into the lamina propria of the matrix compared to untreated matrix. (Fig 14). The authors believe that treated scaffold might be more suitable for bladder tissue engineering reconstruction. In order to enhance the vascularization of tissue engineered bladder scaffold in vitro, Baumert et al. transferred the compound matrix into the greater omentum, which has been mentioned above (21).

After meticulous investigation in vitro, Yoo, et al. used scaffold seeded with multiple cell types to reconstruct bladder tissue in 10 beagle dogs, which on which a partial cystectomy had been performed. As a result, 99% a increased in capacity was achieved in the reconstructed bladder. Immunocytochemical analyses confirmed the urothelial and muscle cell phenotypes and showed the presence of nerve fibers (36). Compared to the the technique of seeding with stromal cells, mesenchymal stem cell-seeded scaffold is becoming much more popular in bladder reconstruction. Chung et al. first performed bladder reconstruction using a BMSCs-seeded SIS in rats. At the level of gene expression, regenerated bladder was similar to the control bladder (37). Compared with the BMSCs, ADSCs can be procured more easily. Therefore, we chose ADSCs-seeded scaffolds for bladder reconstruction. At the end of 24 weeks after the operation, the reconstructed bladders reached a mean volume of 94.68±3.31% of the pre-cystectomy bladder capacity in our study. Smooth muscle cells, urothelium and nerve bundles could be detected by

the original tissue. In the clinic, patients provided with engineered bladder tissue have obtained satisfactory results.

#### **3.1.1 Animal experiments**

Since 1955, many investigators have tried to use different kinds of scaffold for bladder reconstruction in animal models; these have included polyvinyl sponges, polyethylene moulds, Teflon, gelatin sponges, and decellularized pericardial tissue. The outcomes in most studies were unsatisfactory (30). One really successful experiment was reported by Kropp BP et al. (31) in 1995. In this study, the rat underwent partial cystectomy with immediate bladder augmentation with SIS. Host cellular infiltration into the scaffold could be seen 2 weeks after operation. By the end of 48 weeks, the SIS graft presented the three-layered structure of normal bladder, which was indistinguishable from the original bladder. This preliminary study demonstrated the feasibility of using an optimal tissue engineering scaffold for bladder reconstruction. Further study has shown that muscarinic, purinergic and functional cholinergic innervation occurred in rats (32). More recently, bladder regeneration has shown to be more reliable when the SIS was derived from the distal ileum (33). However, graft contraction could be observed in large animal models after using SIS for bladder augmentation, which means that pre-seeding cells may be necessary for tissue engineering-based bladder reconstruction in humans.

Zhang et al. first seeded human bladder urothelial cells and smooth muscle cells onto the SIS by a sandwich culture method (34). This kind of seeding method resulted in organized cell sorting, formation of a well-defined pseudostratified urothelium and multilayered smooth muscle cells with enhanced matrix penetration (Fig 12). The initial study demonstrated that using SIS combined with cell culture could be a valuable model for the study of tissue engineering in bladder reconstruction. To address the problem of graft contraction, Brown et al. showed that opposite-side co-culture of smooth muscle cells and epithelial cells produced a less pronounced matrix contraction than same-side co-culture (20) (Fig 13). The other problem to be addressed in tissue engineering bladder in vitro is cellular infiltration. Recently, Liu et al used preacetic acid (PAA) and Triton X-100

Fig. 12. Sandwich coculture at 28 days shows similar growth pattern to layered coculture technique except that urothelial cells and smooth muscle cells are on opposite sides of small intestinal submucosa membrane. Pseudostratified layer of urothelium is on mucosal surface (open arrow) while multiple layers of smooth muscle cells are on serosal surface and are penetrating into matrix of small intestinal submucosa membrane (solid arrow)(ref 34)

the original tissue. In the clinic, patients provided with engineered bladder tissue have

Since 1955, many investigators have tried to use different kinds of scaffold for bladder reconstruction in animal models; these have included polyvinyl sponges, polyethylene moulds, Teflon, gelatin sponges, and decellularized pericardial tissue. The outcomes in most studies were unsatisfactory (30). One really successful experiment was reported by Kropp BP et al. (31) in 1995. In this study, the rat underwent partial cystectomy with immediate bladder augmentation with SIS. Host cellular infiltration into the scaffold could be seen 2 weeks after operation. By the end of 48 weeks, the SIS graft presented the three-layered structure of normal bladder, which was indistinguishable from the original bladder. This preliminary study demonstrated the feasibility of using an optimal tissue engineering scaffold for bladder reconstruction. Further study has shown that muscarinic, purinergic and functional cholinergic innervation occurred in rats (32). More recently, bladder regeneration has shown to be more reliable when the SIS was derived from the distal ileum (33). However, graft contraction could be observed in large animal models after using SIS for bladder augmentation, which means that pre-seeding cells may be necessary for tissue

Zhang et al. first seeded human bladder urothelial cells and smooth muscle cells onto the SIS by a sandwich culture method (34). This kind of seeding method resulted in organized cell sorting, formation of a well-defined pseudostratified urothelium and multilayered smooth muscle cells with enhanced matrix penetration (Fig 12). The initial study demonstrated that using SIS combined with cell culture could be a valuable model for the study of tissue engineering in bladder reconstruction. To address the problem of graft contraction, Brown et al. showed that opposite-side co-culture of smooth muscle cells and epithelial cells produced a less pronounced matrix contraction than same-side co-culture (20) (Fig 13). The other problem to be addressed in tissue engineering bladder in vitro is

cellular infiltration. Recently, Liu et al used preacetic acid (PAA) and Triton X-100

Fig. 12. Sandwich coculture at 28 days shows similar growth pattern to layered coculture technique except that urothelial cells and smooth muscle cells are on opposite sides of small intestinal submucosa membrane. Pseudostratified layer of urothelium is on mucosal surface (open arrow) while multiple layers of smooth muscle cells are on serosal surface and are penetrating into matrix of small intestinal submucosa membrane (solid arrow)(ref 34)

obtained satisfactory results.

**3.1.1 Animal experiments** 

engineering-based bladder reconstruction in humans.

Fig. 13. Gross appearance of cell seeded constructs at 7 and 28 days post seeding. (A) Collagen Gels, 7 days. (B) BAM, 7days. (C) Collagen Gels, 28 days. (D) BAM, 28 days. Group A: SMCs only. Group C: same side co-culture. Group D: opposite side co-culture (ref 20)

to treat the acelluar matrix for bladder tissue engineering reconstruction in vitro (35). This method led to high porosity on the surface of the matrix with about 75% of normal strength. After 3-D dynamic culture, cells could penetrate deeper into the lamina propria of the matrix compared to untreated matrix. (Fig 14). The authors believe that treated scaffold might be more suitable for bladder tissue engineering reconstruction. In order to enhance the vascularization of tissue engineered bladder scaffold in vitro, Baumert et al. transferred the compound matrix into the greater omentum, which has been mentioned above (21).

After meticulous investigation in vitro, Yoo, et al. used scaffold seeded with multiple cell types to reconstruct bladder tissue in 10 beagle dogs, which on which a partial cystectomy had been performed. As a result, 99% a increased in capacity was achieved in the reconstructed bladder. Immunocytochemical analyses confirmed the urothelial and muscle cell phenotypes and showed the presence of nerve fibers (36). Compared to the the technique of seeding with stromal cells, mesenchymal stem cell-seeded scaffold is becoming much more popular in bladder reconstruction. Chung et al. first performed bladder reconstruction using a BMSCs-seeded SIS in rats. At the level of gene expression, regenerated bladder was similar to the control bladder (37). Compared with the BMSCs, ADSCs can be procured more easily. Therefore, we chose ADSCs-seeded scaffolds for bladder reconstruction. At the end of 24 weeks after the operation, the reconstructed bladders reached a mean volume of 94.68±3.31% of the pre-cystectomy bladder capacity in our study. Smooth muscle cells, urothelium and nerve bundles could be detected by

Tissue Engineering in Low Urinary Tract Reconstruction 439

Fig. 15. Cystographies of bladders reconstructed 4 weeks postoperatively. a The control group, b the experimental group 24 weeks postoperatively, c the experimental group and d the experimental group. Cystography demonstrated an improvement in both the shape

Fig. 16. Histological features of the transplanted grafts. Four weeks postoperatively, native bladder tissue (blue arrow) and in the graft (yellow arrow). a The control group, b the

and capacity of bladders reconstructed with seeded matrices

Fig. 14. Cell penetration in 5% PAA-treated BSM in different speeds (0, 10 and 40 rpm) in three-dimensional dynamic culture. Smooth muscle cells and urothelial cell were seeded as layers and co-cultured on the submucosa side of 5% PAA-treated BSM using static culture (left column; a,b) and 3-D rotation culture conditions at 10 (middle column; c,d) and 40 rpm (right column; e,f). H&E staining (a,c,e) and DAPI staining (b,d,f) are shown at 200. Compared with static culture (left column), the cells grew uniformly with deeper penetration in the matrix using 3-D dynamic culture (middle and right column). The cells grown at 40 rpm had deeper penetration of cells within the matrix (e,f) compared to cells cultured at 10 rpm rotation speed (c,d) (ref 35)

immunohistochemical assays. On the contrary, the mean bladder volume was 69.33±5.05% in the control group, made using unseeded scaffolds, and in these there was no evidence of organized muscle or nerve tissue (10) (Fig 15,16). In our study, we also noted that the optimal area for bladder regeneration using seeded scaffold is more than 40-60%, since smaller areas can be regenerated by native bladder tissue. These data provide a useful reference for further clinical application. Other stem cell-seeded scaffolds have also been reported for bladder reconstruction, such as hair-follicle stem cell-seeded scaffolds and urine-derived stem cell-seeded scaffolds (12). However, the number of reports is limited and the actual effectiveness of those scaffold need to be further studied.

As well as traditional cell seeded scaffolds, many modification techniques have been used for tissue engineered bladder reconstructions in animal models. Gregory et al. seeded human adipose stem cells onto PLGA (85:15) bladder dome composites and grafted the result into rat hosts. Results showed that bladder capacity and compliance were maintained in the cell-seeded group throughout the 12 weeks (38) (Fig 17). SIS, modified by hyaluronic acid nanoparticles, has been used for bladder reconstruction. Urinary bladder augmentation has been performed in beagle dogs following hemi-cystectomy using nanoparticle-modified SIS. The results showed that the modified scaffold had significantly higher vascularity compared to unmodified one. This report demonstrated that the nanotechnology can represent a new approach for modifying biomaterials in bladder reconstruction (39). Wei et al.

Fig. 14. Cell penetration in 5% PAA-treated BSM in different speeds (0, 10 and 40 rpm) in three-dimensional dynamic culture. Smooth muscle cells and urothelial cell were seeded as layers and co-cultured on the submucosa side of 5% PAA-treated BSM using static culture (left column; a,b) and 3-D rotation culture conditions at 10 (middle column; c,d) and 40 rpm

penetration in the matrix using 3-D dynamic culture (middle and right column). The cells grown at 40 rpm had deeper penetration of cells within the matrix (e,f) compared to cells

immunohistochemical assays. On the contrary, the mean bladder volume was 69.33±5.05% in the control group, made using unseeded scaffolds, and in these there was no evidence of organized muscle or nerve tissue (10) (Fig 15,16). In our study, we also noted that the optimal area for bladder regeneration using seeded scaffold is more than 40-60%, since smaller areas can be regenerated by native bladder tissue. These data provide a useful reference for further clinical application. Other stem cell-seeded scaffolds have also been reported for bladder reconstruction, such as hair-follicle stem cell-seeded scaffolds and urine-derived stem cell-seeded scaffolds (12). However, the number of reports is limited and

As well as traditional cell seeded scaffolds, many modification techniques have been used for tissue engineered bladder reconstructions in animal models. Gregory et al. seeded human adipose stem cells onto PLGA (85:15) bladder dome composites and grafted the result into rat hosts. Results showed that bladder capacity and compliance were maintained in the cell-seeded group throughout the 12 weeks (38) (Fig 17). SIS, modified by hyaluronic acid nanoparticles, has been used for bladder reconstruction. Urinary bladder augmentation has been performed in beagle dogs following hemi-cystectomy using nanoparticle-modified SIS. The results showed that the modified scaffold had significantly higher vascularity compared to unmodified one. This report demonstrated that the nanotechnology can represent a new approach for modifying biomaterials in bladder reconstruction (39). Wei et al.

(right column; e,f). H&E staining (a,c,e) and DAPI staining (b,d,f) are shown at 200. Compared with static culture (left column), the cells grew uniformly with deeper

the actual effectiveness of those scaffold need to be further studied.

cultured at 10 rpm rotation speed (c,d) (ref 35)

Fig. 15. Cystographies of bladders reconstructed 4 weeks postoperatively. a The control group, b the experimental group 24 weeks postoperatively, c the experimental group and d the experimental group. Cystography demonstrated an improvement in both the shape and capacity of bladders reconstructed with seeded matrices

Fig. 16. Histological features of the transplanted grafts. Four weeks postoperatively, native bladder tissue (blue arrow) and in the graft (yellow arrow). a The control group, b the

Tissue Engineering in Low Urinary Tract Reconstruction 441

Fig. 18. Construction of engineered bladder scaffold seeded with cells (A) and engineered bladder anastamosed to native bladder with running 4–0 polyglycolic sutures (B). Implant

Fig. 19. Preoperative (A) and 10-months postoperative (B) cystograms and urodynamic

In another clinical study reported in the 2008 AUA, patients, who had received tissue engineered bladder showed increasing capacity and reduced intravesicular pressure. According to these reports, there is a clear reason to hope that the tissue engineered bladder can be utilized for a fully functioning neurogenic bladder. More indications about using this

The application of tissue engineering techniques for urethral reconstruction has been developed in recent years and the potential market for a tissue engineered solution for

ndings in patient with a collagen-PGA scaff old engineered bladder (Ref 40)

urethral stricture and abnormality will continue to increase in the near future.

kind of biomaterial might be obtained in the near future.

**3.2 Urethral reconstruction** 

covered with brin glue and omentum (C) (Ref 40)

experimental group 24 weeks postoperatively, c in the control group there is no evidence of organized bladder tissue regeneration, d in the experimental group, the grafts had formed a multilayer epithelium with organized smooth muscle cells. Immunohistochemistry of the transplanted grafts. e Staining with cytokeratin AE1/AE3. -SM actin. g S-100 (arrows).

Fig. 17. Construction of the three dimensional synthetic bladder composite. a: Schematic and b: gross micrograph of the three dimensional bladder composite. c:PLGA electropulled microbers comprising the luminal layer. d: PLGA porous sponge was used as the outer layer (ref 38).

designed a bioreactor to simulate the mechanical properties of bladder. This system successfully generated appropriate pressure waveforms. The viability of cells and tissue structures observed after culture in simulated conditions showed that mechanical stimulation improved the arrangement of cells on scaffold (24).

#### **3.1.2 Clinical application**

Although the reports about bladder reconstruction using tissue engineering techniques are few, they are the landmarks in tissue engineered lower urinary tract reconstruction. In Atala's famous study, seven patients with myelomeningocele with high-pressure or poorly compliant bladders enrolled. Urothelial and muscle cells were seeded on a biodegradable bladder-shaped scaffold made of collagen and PGA. Then the biomaterial was used for reconstruction with an omental wrap (40). (Fig 18) After the operation, none of the ultrasounds showed any abnormalities. The cystogram showed the regular shape of bladder after the reconstruction. Urodynamic studies demonstrated significant improvement in volume and compliance in the composite engineered bladders (Fig 19). Postoperatively, it is difficult to distinguish the margin between the composite matrix and the native bladders grossly. All biopsies showed a trilayered structure, consisting of a urothelial cell-lined lumen surrounded by submucosa and muscle. During the post-operative follow-up period all patients had a stable renal function in which serum creatinine was similar to the preoperative status. No metabolic abnormalities were noted. There was no evidence of urinary calculi during the study.

experimental group 24 weeks postoperatively, c in the control group there is no evidence of organized bladder tissue regeneration, d in the experimental group, the grafts had formed a multilayer epithelium with organized smooth muscle cells. Immunohistochemistry of the transplanted grafts. e Staining with cytokeratin AE1/AE3. -SM actin. g S-100 (arrows).

Fig. 17. Construction of the three dimensional synthetic bladder composite. a: Schematic and b: gross micrograph of the three dimensional bladder composite. c:PLGA electropulled microbers comprising the luminal layer. d: PLGA porous sponge was used as the outer

designed a bioreactor to simulate the mechanical properties of bladder. This system successfully generated appropriate pressure waveforms. The viability of cells and tissue structures observed after culture in simulated conditions showed that mechanical

Although the reports about bladder reconstruction using tissue engineering techniques are few, they are the landmarks in tissue engineered lower urinary tract reconstruction. In Atala's famous study, seven patients with myelomeningocele with high-pressure or poorly compliant bladders enrolled. Urothelial and muscle cells were seeded on a biodegradable bladder-shaped scaffold made of collagen and PGA. Then the biomaterial was used for reconstruction with an omental wrap (40). (Fig 18) After the operation, none of the ultrasounds showed any abnormalities. The cystogram showed the regular shape of bladder after the reconstruction. Urodynamic studies demonstrated significant improvement in volume and compliance in the composite engineered bladders (Fig 19). Postoperatively, it is difficult to distinguish the margin between the composite matrix and the native bladders grossly. All biopsies showed a trilayered structure, consisting of a urothelial cell-lined lumen surrounded by submucosa and muscle. During the post-operative follow-up period all patients had a stable renal function in which serum creatinine was similar to the preoperative status. No metabolic abnormalities were noted. There was no evidence of

stimulation improved the arrangement of cells on scaffold (24).

layer (ref 38).

**3.1.2 Clinical application** 

urinary calculi during the study.

Fig. 18. Construction of engineered bladder scaffold seeded with cells (A) and engineered bladder anastamosed to native bladder with running 4–0 polyglycolic sutures (B). Implant covered with brin glue and omentum (C) (Ref 40)

Fig. 19. Preoperative (A) and 10-months postoperative (B) cystograms and urodynamic ndings in patient with a collagen-PGA scaff old engineered bladder (Ref 40)

In another clinical study reported in the 2008 AUA, patients, who had received tissue engineered bladder showed increasing capacity and reduced intravesicular pressure. According to these reports, there is a clear reason to hope that the tissue engineered bladder can be utilized for a fully functioning neurogenic bladder. More indications about using this kind of biomaterial might be obtained in the near future.

#### **3.2 Urethral reconstruction**

The application of tissue engineering techniques for urethral reconstruction has been developed in recent years and the potential market for a tissue engineered solution for urethral stricture and abnormality will continue to increase in the near future.

Tissue Engineering in Low Urinary Tract Reconstruction 443

Fig. 20. buccal mucosa culture at a, the air–liquid interface; b, submerged; c, at day 1 ALI; d, at day 5 ALI; e, at day 8 ALI and with f, Protocol 2 (cells on same surface) (Ref 44)

Fig. 21. Histological results of tissue engineering urethral after reconstruction. a. tissue engineering urethra using foreskin seeded scaffold; B, tissue engineering urethra using oral

(Group C), 6 scaffolds with only lingual keratinocytes (Group B) and 6 matrices without cells (Group A) were used to repair a rabbit urethral defect. H&E staining of seeded ACSM showed several epithelial layers and well distributed CSMCs in the matrix. The maintenance of wide urethral caliber could be observed in Group C, while strictures were observed in groups A and B (Fig 22). Histologically, the retrieved urethra in group A showed fibrosis and inflammation during 6 months. A simple epithelial layer regenerated in group B but there was still no evidence of CSMCs growing into grafts during study period. A stratified epithelial layer and organized muscle fiber bundles were evident 6 months after implantation in group C (Fig 23). Our results demonstrated that lingual keratinocytes and CSMCs could be used as a source of seed cells for urethral tissue engineering. Using the

keratinocytes seeded scaffold. (Ref 44,45)

#### **3.2.1 Animal experiments**

In animal experiments addressing urethral reconstruction, the first attempts used biomaterials alone. Among these experiments, most papers reported the application of SIS in animal urethroplasty. Many results were encouraging. Regenerated urethra contained a well-differentiated epithelium, underneath which was circular smooth muscle and abundant collagen and fibrous connective tissue. The only difference between the SISreconstructed urethra and normal urethra was the amount and size of the circular bundles of smooth muscle. However, several key points should be considered before using this kind of biomaterial. First, El-Assmy mentioned that locally prepared SIS and commercially available SIS may lead to the different results (41).This might be related to different pore sizes, which limit the infiltration and migration of cells. Second, the feasibility of using tabularized matrix for urethroplasty is still controversy although SIS has been proven to be useful for onlay urethroplasty. In Shokeir's study, a 3cm segment of the whole urethral circumference was excised and replaced by a tube matrix of the same length and width in 14 dogs. However, all dogs suffered a urethral fistula and/or stricture after the stent removal. This result demonstrated that a tube formed of matrix without seeded cells was not able to replace the long segment including the whole circumference of the canine urethra (42). Third, the length of urethral defect is another key point that should be considered during the urethroplasty. In order to investigate the maximum distance for normal tissue regeneration, Dorin et al. performed the tabularized urethroplasty in 12 male rabbits using acellular scaffold at varying lengths (0.5,1,2 and 3cm). The final result indicated that the maximal defect distance suitable for normal tissue formation using acellular grafts that rely on the native cells for tissue regeneration appears to be 0.5cm (43). Although other reports showed that the synthetic scaffold alone could be used for urethroplasty, the need ror a move to using cell-seeded scaffold is obvious.

In 2003, Bhargava has developed tissue-engineered buccal mucosa for use in substitution urethroplasty. Histologically, the matrix closely resembled the native oral mucosa after culturing for 2 weeks. A gradually increasing thickness of the epidermis and remodeling of the dermis could also be seen (44) (Fig 20). Subsequently, more cell-seeded scaffolds were used for urethral reconstruction in our center. Li et al. replaced urinary epithelial cells with oral keratinocytes seeded on BAMG to reconstruct a tissue-engineered urethra. Histological results showed that multiple layers of keratinocytes had formed at 2 and 6 months after the operation. Obvious margins between graft oral keratinocytes and host epithelium could be noticed in H&E sections (Fig 21b). Fu et al. used foreskin epidermal cell-seeded scaffolds to repair a urethra defect in a rabbit model. During following up, several layers of epidermal cells with abundant vessels in the submucosa were noticed. Moreover, immunofluorescence confirmed the survival of implanted epidermal cells at 1 month after procedure (45,46) (Fig 21a).

Recently, we have investigated the feasibility of constructing 3D structure urethra using multiple seeding cell types. It has also been hypothesized that building three-dimensional constructs in vitro prior to implantation would facilitate matrix vascularization in vivo and minimize the inflammatory response towards the matrix. Therefore, we seeded autologus corporal smooth muscle cells (CSMCs) and lingual keratinocytes into ACSM, using a staticdynamic seeding method. After being cultured 14 days, 6 scaffolds with two kind of cells

In animal experiments addressing urethral reconstruction, the first attempts used biomaterials alone. Among these experiments, most papers reported the application of SIS in animal urethroplasty. Many results were encouraging. Regenerated urethra contained a well-differentiated epithelium, underneath which was circular smooth muscle and abundant collagen and fibrous connective tissue. The only difference between the SISreconstructed urethra and normal urethra was the amount and size of the circular bundles of smooth muscle. However, several key points should be considered before using this kind of biomaterial. First, El-Assmy mentioned that locally prepared SIS and commercially available SIS may lead to the different results (41).This might be related to different pore sizes, which limit the infiltration and migration of cells. Second, the feasibility of using tabularized matrix for urethroplasty is still controversy although SIS has been proven to be useful for onlay urethroplasty. In Shokeir's study, a 3cm segment of the whole urethral circumference was excised and replaced by a tube matrix of the same length and width in 14 dogs. However, all dogs suffered a urethral fistula and/or stricture after the stent removal. This result demonstrated that a tube formed of matrix without seeded cells was not able to replace the long segment including the whole circumference of the canine urethra (42). Third, the length of urethral defect is another key point that should be considered during the urethroplasty. In order to investigate the maximum distance for normal tissue regeneration, Dorin et al. performed the tabularized urethroplasty in 12 male rabbits using acellular scaffold at varying lengths (0.5,1,2 and 3cm). The final result indicated that the maximal defect distance suitable for normal tissue formation using acellular grafts that rely on the native cells for tissue regeneration appears to be 0.5cm (43). Although other reports showed that the synthetic scaffold alone could be used for urethroplasty, the need ror a

In 2003, Bhargava has developed tissue-engineered buccal mucosa for use in substitution urethroplasty. Histologically, the matrix closely resembled the native oral mucosa after culturing for 2 weeks. A gradually increasing thickness of the epidermis and remodeling of the dermis could also be seen (44) (Fig 20). Subsequently, more cell-seeded scaffolds were used for urethral reconstruction in our center. Li et al. replaced urinary epithelial cells with oral keratinocytes seeded on BAMG to reconstruct a tissue-engineered urethra. Histological results showed that multiple layers of keratinocytes had formed at 2 and 6 months after the operation. Obvious margins between graft oral keratinocytes and host epithelium could be noticed in H&E sections (Fig 21b). Fu et al. used foreskin epidermal cell-seeded scaffolds to repair a urethra defect in a rabbit model. During following up, several layers of epidermal cells with abundant vessels in the submucosa were noticed. Moreover, immunofluorescence confirmed the survival of implanted epidermal cells at 1

Recently, we have investigated the feasibility of constructing 3D structure urethra using multiple seeding cell types. It has also been hypothesized that building three-dimensional constructs in vitro prior to implantation would facilitate matrix vascularization in vivo and minimize the inflammatory response towards the matrix. Therefore, we seeded autologus corporal smooth muscle cells (CSMCs) and lingual keratinocytes into ACSM, using a staticdynamic seeding method. After being cultured 14 days, 6 scaffolds with two kind of cells

**3.2.1 Animal experiments** 

move to using cell-seeded scaffold is obvious.

month after procedure (45,46) (Fig 21a).

Fig. 20. buccal mucosa culture at a, the air–liquid interface; b, submerged; c, at day 1 ALI; d, at day 5 ALI; e, at day 8 ALI and with f, Protocol 2 (cells on same surface) (Ref 44)

Fig. 21. Histological results of tissue engineering urethral after reconstruction. a. tissue engineering urethra using foreskin seeded scaffold; B, tissue engineering urethra using oral keratinocytes seeded scaffold. (Ref 44,45)

(Group C), 6 scaffolds with only lingual keratinocytes (Group B) and 6 matrices without cells (Group A) were used to repair a rabbit urethral defect. H&E staining of seeded ACSM showed several epithelial layers and well distributed CSMCs in the matrix. The maintenance of wide urethral caliber could be observed in Group C, while strictures were observed in groups A and B (Fig 22). Histologically, the retrieved urethra in group A showed fibrosis and inflammation during 6 months. A simple epithelial layer regenerated in group B but there was still no evidence of CSMCs growing into grafts during study period. A stratified epithelial layer and organized muscle fiber bundles were evident 6 months after implantation in group C (Fig 23). Our results demonstrated that lingual keratinocytes and CSMCs could be used as a source of seed cells for urethral tissue engineering. Using the

Tissue Engineering in Low Urinary Tract Reconstruction 445

Fig. 24. a. Tubular tissue was gently everted so that the mesothelium lined the lumen. b The

According to this result, the authors concluded that autologous tissue grown within the recipients' peritoneal cavity can be used successfully for tabularized urethral reconstruction (48). In addition, synthetic matrix combined with seeded cells has also been used for urethroplasty. In Selim's study, the optimal sterilization and cell seeding method for synthetic biomaterials in urethral reconstruction has been investigated. In their study, both PAA and γ-irradiation appear to be suitable methods for sterilizing PLGA scaffolds. And the sterilized PLGA 85:15 is a promising material for tissue engineering urethral

Up till now, many urologists have reported successful outcomes of urethral reconstruction using tissue engineering techniques. Most reports have been focused on treating urethral strictures using SIS. Among them, most results were satisfactory (Table 2) (50-56). In the report of Fiala et al., fifty patients with urethral strictures received urethroplasty using SIS. During post-operative follow-up, clinical, radiological, and cosmetic findings were excellent in 80% patients. No complications, such as fistulae, wound infections, or rejection were observed. This is so far the largest reports about using SIS for urethral reconstruction, in terms of numbers of patients. Their results were more satisfacory even than traditional urethral reconstruction using buccal or lingual mucosa for such low complication rate. In our center, we have also used SIS patch to undergo in 16 male patients with urethral strictures. The average length of strictures was 4.6 cm, ranging from 3.5-6 cm (Fig 25). After the operation, urethrography showed a wide patent urethra in all patients. The mean Qmax increased obviously from 3.8ml/s to 25ml/s. Only one patient needed urethral dilation due to the decreasing of Qmax at the end of 5 months. During follow-up, routine urethroscopy was performed in all patients. At the end of 4 weeks after operation, SIS could be easily noticed in the urethral lumen. However, the implanted graft could not be identified from the normal urethra 38 weeks after operation (Fig 26). The HE staining of biopsy showed that

everted tubular tissue was interposed and anastomosed as urethral graft (Ref 48)

strictures could be observed by the end of 6 months. Meanwhile, multilayer squamous

epithelial layers covered the surface of the urethra.

reconstruction (49).

**3.2.2 Clinical application** 

dynamic-static seeding method, a 3-D urethra could be constructed in vivo. It can provide us an alternative method to treat the urethral disease using tissue engineering techniquea.

Fig. 22. Comparision of urethrography image in each group at 1,2,6 month after operation. The arrow indicates the stricture site of urethra

Fig. 23. Macroscopic inspection and H&E staining (inset) of retrieved urethrae in each group at 1,2,6 month after operation. In group A, a urethral stricture existed at every study time point. H&E staining did not show continuous epithelial layers but did show severe inflammation. In group B, strictures could be noticed by gross inspection. Only 1-2 epithelial layers were formed at 6 months after implantation. In group C, patent lumens without

dynamic-static seeding method, a 3-D urethra could be constructed in vivo. It can provide us an alternative method to treat the urethral disease using tissue engineering techniquea.

Fig. 22. Comparision of urethrography image in each group at 1,2,6 month after operation.

Fig. 23. Macroscopic inspection and H&E staining (inset) of retrieved urethrae in each group at 1,2,6 month after operation. In group A, a urethral stricture existed at every study time point. H&E staining did not show continuous epithelial layers but did show severe

inflammation. In group B, strictures could be noticed by gross inspection. Only 1-2 epithelial layers were formed at 6 months after implantation. In group C, patent lumens without

The arrow indicates the stricture site of urethra

strictures could be observed by the end of 6 months. Meanwhile, multilayer squamous epithelial layers covered the surface of the urethra.

Fig. 24. a. Tubular tissue was gently everted so that the mesothelium lined the lumen. b The everted tubular tissue was interposed and anastomosed as urethral graft (Ref 48)

According to this result, the authors concluded that autologous tissue grown within the recipients' peritoneal cavity can be used successfully for tabularized urethral reconstruction (48). In addition, synthetic matrix combined with seeded cells has also been used for urethroplasty. In Selim's study, the optimal sterilization and cell seeding method for synthetic biomaterials in urethral reconstruction has been investigated. In their study, both PAA and γ-irradiation appear to be suitable methods for sterilizing PLGA scaffolds. And the sterilized PLGA 85:15 is a promising material for tissue engineering urethral reconstruction (49).

#### **3.2.2 Clinical application**

Up till now, many urologists have reported successful outcomes of urethral reconstruction using tissue engineering techniques. Most reports have been focused on treating urethral strictures using SIS. Among them, most results were satisfactory (Table 2) (50-56). In the report of Fiala et al., fifty patients with urethral strictures received urethroplasty using SIS. During post-operative follow-up, clinical, radiological, and cosmetic findings were excellent in 80% patients. No complications, such as fistulae, wound infections, or rejection were observed. This is so far the largest reports about using SIS for urethral reconstruction, in terms of numbers of patients. Their results were more satisfacory even than traditional urethral reconstruction using buccal or lingual mucosa for such low complication rate. In our center, we have also used SIS patch to undergo in 16 male patients with urethral strictures. The average length of strictures was 4.6 cm, ranging from 3.5-6 cm (Fig 25). After the operation, urethrography showed a wide patent urethra in all patients. The mean Qmax increased obviously from 3.8ml/s to 25ml/s. Only one patient needed urethral dilation due to the decreasing of Qmax at the end of 5 months. During follow-up, routine urethroscopy was performed in all patients. At the end of 4 weeks after operation, SIS could be easily noticed in the urethral lumen. However, the implanted graft could not be identified from the normal urethra 38 weeks after operation (Fig 26). The HE staining of biopsy showed that

Tissue Engineering in Low Urinary Tract Reconstruction 447

Fig. 26. Urethroscopy after the urethral reconstruction using SIS graft.Arrow headed the implanted site. a:4 weeks after op; b: 6 weeks after op; c: 38 weeks after operation.

Fig. 27. The HE staining of biopsy showed that stratified squamous epithelial layers have

Cell-seeded scaffolds have also been used for urethral reconstruction in some patients. Based on the previous reports mentioned above, Bhargava et al. useed autologous tissueengineered buccal mucosa to treat five patients with urethral strictures secondary to lichen sclerosis. After the intimal operation, one patient had complete excision of the grafted urethra and one required partial graft excision. The other three patients required some form of instrumentation although endoscopic appearance showed a patent urethra with the implanted graft in situ (57). Recently, Atala et al. reported that using cell-seeded synthetic tubularized scaffolds to repair urethral defects in five boys. At the end of follow-up, some satisfactory results were obtained. The median end Qmax was 27.1 ml/s, and serial urethrographic and endoscopic studies showed the maintenance of wide calibres without

grown on the SIS implanted site.

strictures (Fig 28) (58).

stratified squamous epithelial layers had grown on the SIS implanted site, which was similar to normal urethral mucosa (Fig 27). According to these clinical experiences, the use of an acellular matrix SIS for urethroplasty should only be done when the length of urethral stricture is short. Patients with a bulbar urethral stricture are more suitable than those with a urethral stricture in other sites. Of course, the condition of urethral plate should also be considered before using SIS. We believe that urethroplasty using tissue engineered scaffold can achieve a satisfactory outcome that is similar to the gold-standard procedure *provided optimal patients are selected*.


Table 2. Recoder of using SIS for urethral reconstruction in clinic

Fig. 25. Application of SIS in urethroplasty. a. penile urethral stricture; b.bulbopenile urethral stricture; c.bulbar urethral stricture.

stratified squamous epithelial layers had grown on the SIS implanted site, which was similar to normal urethral mucosa (Fig 27). According to these clinical experiences, the use of an acellular matrix SIS for urethroplasty should only be done when the length of urethral stricture is short. Patients with a bulbar urethral stricture are more suitable than those with a urethral stricture in other sites. Of course, the condition of urethral plate should also be considered before using SIS. We believe that urethroplasty using tissue engineered scaffold can achieve a satisfactory outcome that is similar to the gold-standard procedure *provided* 

Table 2. Recoder of using SIS for urethral reconstruction in clinic

Fig. 25. Application of SIS in urethroplasty.

a. penile urethral stricture; b.bulbopenile urethral stricture; c.bulbar urethral stricture.

*optimal patients are selected*.

Fig. 26. Urethroscopy after the urethral reconstruction using SIS graft.Arrow headed the implanted site. a:4 weeks after op; b: 6 weeks after op; c: 38 weeks after operation.

Fig. 27. The HE staining of biopsy showed that stratified squamous epithelial layers have grown on the SIS implanted site.

Cell-seeded scaffolds have also been used for urethral reconstruction in some patients. Based on the previous reports mentioned above, Bhargava et al. useed autologous tissueengineered buccal mucosa to treat five patients with urethral strictures secondary to lichen sclerosis. After the intimal operation, one patient had complete excision of the grafted urethra and one required partial graft excision. The other three patients required some form of instrumentation although endoscopic appearance showed a patent urethra with the implanted graft in situ (57). Recently, Atala et al. reported that using cell-seeded synthetic tubularized scaffolds to repair urethral defects in five boys. At the end of follow-up, some satisfactory results were obtained. The median end Qmax was 27.1 ml/s, and serial urethrographic and endoscopic studies showed the maintenance of wide calibres without strictures (Fig 28) (58).

Tissue Engineering in Low Urinary Tract Reconstruction 449

[4] Fu Q, Deng CL, Song XF, Xu YM. Long-term study of male rabbit urethral mucosa

[5] Kimball JR, Nittayananta W, Klausner M, Chung WO, Dale BA. Antimicrobial barrier of

[6] Li C, Xu Y, Song L, Fu Q, Cui L, Yin S. Preliminary experimental study of tissue-

[7] Rajasekaran M, Kasyan A, Allilain W, Monga M. Ex vivo expression of angiogenic

[8] Drzewiecki BA, Thomas JC, Tanaka ST. Bone marrow-derived mesenchymal stem cells:

[9] Zhang Y, Lin HK, Frimberger D, Epstein RB, Kropp BP. Growth of bone marrow stromal

[10] Zhu WD, Xu YM, Feng C, Fu Q, Song LJ, Cui L. Bladder reconstruction with adipose-

[11] Tian H, Bharadwaj S, Liu Y, Ma PX, Atala A, Zhang Y. Differentiation of human bone

[12] Drewa T, Joachimiak R, Kaznica A, Sarafian V, Pokrywczynska M. Hair stem cells for

[13] Zhang Y, McNeill E, Tian H, Soker S, Andersson KE, Yoo JJ, et al. Urine derived cells

[14] Bodin A, Bharadwaj S, Wu S, Gatenholm P, Atala A, Zhang Y. Tissue-engineered

[16] Brehmer B, Rohrmann D, Becker C, Rau G, Jakse G. Different types of scaffolds for reconstruction of the urinary tract by tissue engineering. Urol Int 2007;78:23–29. [17] Feng C, Xu YM, Fu Q, Zhu WD, Cui L, Chen J. Evaluation of the biocompatibility and

[18] Freytes DO, Badylak SF, Webster TJ, Geddes LA, Rundell AE. Biaxial strength of

reconstruction. J Biomed Mater Res A. 2010;94:317-325.

urinary reconstruction and diversion. Biomaterials. 2010;31:8889-8901. [15] Nakanishi Y, Chen G, Komuro H, Ushida T, Kaneko S, Tateishi T,Kaneko M. Tissue-

engineered urethral reconstruction using oral keratinocytes seeded on BAMG. Urol

growth factors and their receptors in human penile cavernosal cells. J Androl.

current and future applications in the urinary bladder. Stem Cells Int. 2011,3; 765-

cells on small intestinal submucosa an alternative cell source for tissue engineered

derived stem cell-seeded bladder acellular matrix grafts improve morphology

marrow mesenchymal stem cells into bladder cells: potential for urological tissue

bladder regeneration in rats: preliminary results. Transplant Proc. 2009;41:4345-

are a potential source for urological tissue reconstruction. J Urol 2008;180:2226-

conduit using urine-derived stem cells seeded bacterial cellulose polymer in

engineered urinary bladder wall using PLGA mesh-collagen hybrid scaffolds: A comparison study of collagen sponge and gel as a scaffold. J Pediatr Surg

mechanical properties of naturally derived and synthetic scaffolds for urethral

multilaminated extracellular matrix scaffolds. Biomaterials. 2004 May;25:2353-

an in vitro oral epithelial model. Arch Oral Biol. 2006;51:775-783

reconstruction. Asian J Androl. 2008;10:719-722.

Int. 2008;81:290-295.

bladder. BJU Int. 2005;96:1120-1125.

composition. World J Urol. 2010;28:493-498.

engineering. Tissue Eng Part A. 2010;16:1769-1779.

2003;24:85-90

767

4351.

2233

2361.

2003;38:1781–1784

Fig. 28. A cell-seeded graft sutured to the normal urethral margins(Ref 59)

#### **4. Challenges and risks**

Based on previous studies, the potential market for a tissue-engineered solution for dysfunctional bladders and small contracted or inflamed bladders is probably far too small for commercial exploitation. The only two potential indications for commercial-scale tissue engineering lower urinary tract tissue are bladder carcinoma and urethral stricture. Cellseeded scaffolds will probably be further investigated and applied in clinics. hreedimensional structures and the use of bioreactors will also be more and more popular in tissue engineering research for lower urinary tract reconstruction.

However, several problems need to be solved. For example, the ethical problems about the implanted matrix (and where it is obtained) needs to be further discussed. The potential for carcinogenic problems arising form the use of stem cells is not clear. Optimal methods of cell labeling (for research) still needs to be improved.

Nevertheless, there is no doubt that tissue engineering techniques for lower urinary tract reconstruction will themselves become the gold-standard in the near future. A substantial commercial market will continue to grow and more patients will obtain benefit from this technique.

#### **5. References**


Fig. 28. A cell-seeded graft sutured to the normal urethral margins(Ref 59)

tissue engineering research for lower urinary tract reconstruction.

labeling (for research) still needs to be improved.

system. Am J Transplant. 2004;4:58-73.

Based on previous studies, the potential market for a tissue-engineered solution for dysfunctional bladders and small contracted or inflamed bladders is probably far too small for commercial exploitation. The only two potential indications for commercial-scale tissue engineering lower urinary tract tissue are bladder carcinoma and urethral stricture. Cellseeded scaffolds will probably be further investigated and applied in clinics. hreedimensional structures and the use of bioreactors will also be more and more popular in

However, several problems need to be solved. For example, the ethical problems about the implanted matrix (and where it is obtained) needs to be further discussed. The potential for carcinogenic problems arising form the use of stem cells is not clear. Optimal methods of cell

Nevertheless, there is no doubt that tissue engineering techniques for lower urinary tract reconstruction will themselves become the gold-standard in the near future. A substantial commercial market will continue to grow and more patients will obtain benefit from this

[1] Atala A. Tissue engineering for the replacement of organ function in the genitourinary

[2] Brown AL, Brook-Allred TT, Waddell JE, White J, Werkmeister JA, Ramshaw JA, et al.

[3] De Filippo RE, Yoo JJ, Atala A. Urethra replacement using cell seeded tabularize collagen matrices. J Urol 2002; 168: 1789–1792; discussion 1792–1793

urothelial cell interactions. Biomaterials. 2005;26:529-543.

Bladder acellular matrix as a substrate for studying in vitr bladder smooth muscle–

**4. Challenges and risks** 

technique.

**5. References** 


Tissue Engineering in Low Urinary Tract Reconstruction 451

[36] Yoo JJ, Meng J, Oberpenning F, Atala A. Bladder augmentation using allogenic bladder

[37] Chung SY, Krivorov NP, Rausei V, Thomas L, Frantzen M, Landsittel D, et al. Bladder

[38] Jack GS, Zhang R, Lee M, Xu Y, Wu BM, Rodríguez LV.Urinary bladder smooth muscle

[39] Mondalek FG, Ashley RA, Roth CC, Kibar Y, Shakir N, Ihnat MA, et al. Enhanced

[40] Atala A, Bauer SB, Soker S, Yoo JJ, Retik AB. Tissue-engineered autologous bladders for

[41] El-Assmy A, El-Hamid MA, Hafez AT.Urethral replacement: a comparison between

[42] Shokeir A, Osman Y, Gabr M, Mohsen T, Dawaba M, el-Baz M.Acellular matrix tube for canine urethral replacement:Is it fact or fiction. J Urol. 2004;171:453-456. [43] Dorin RP, Pohl HG, De Filippo RE, Yoo JJ, Atala A. Tubularized urethral replacement

[44] Bhargava S, Chapple CR, Bullock AJ, Layton C, MacNeil S.Tissue-engineered buccal

[45] Fu Q, Deng CL, Liu W, Cao YL.Urethral replacement using epidermal cell-seeded tubular acellular bladder collagen matrix. BJU Int. 2007;99:1162-1165. [46] Li C, Xu YM, Song LJ, Fu Q, Cui L, Yin S.Urethral reconstruction using oral keratinocyte

[47] Guan Y, Ou L, Hu G, Wang H, Xu Y, Chen J, et al. Tissue engineering of urethra using

[48] Gu GL, Zhu YJ, Xia SJ, Zhang J, Jiang JT, Hong Y, et al. Peritoneal cavity as bioreactor to

[49] Selim M, Bullock AJ, Blackwood KA, Chapple CR, MacNeil S. Developing

[50] Mantovani F, Trinchieri A, Castelnuovo C, Romanò AL, Pisani E. Reconstructive urethroplasty using porcine acellular matrix. Eur Urol. 2003;44:600-602. [51] Le Roux JP. Endoscopic urethroplasty with unseeded small intestinal submucosa

collagen matrix grafts: a pilot study. J Urol. 2005 ;173:140-143.

human vascular endothelial growth factor gene-modied bladder urothelial cells.

grow autologous tubular urethral grafts in a rabbit model. World J

biodegradable scaffolds for tissue engineering of the urethra. BJU Int. 2011;107:296-

mucosa for substitution urethroplasty. BJU Int. 2004;93:807-811.

seeded bladder acellular matrix grafts. J Urol. 2008 ;180:1538-1542.

reconstruction with bone marrow derived stem cells seeded on small intestinal submucosa improves morphological and molecular composition. J

engineered from adipose stem cells and a three dimensional synthetic composite.

angiogenesis of modied porcine small intestinal submucosa with hyaluronic acidpoly(lactide-co-glycolide) nanoparticles: From fabrication to preclinical validation. J

small intestinal submucosa grafts and spontaneous regeneration. BJU

with unseeded matrices: what is the maximum distance for normal tissue

submucosa seeded with cells. Urology. 1998;51:221-225.

patients needing cystoplasty. Lancet. 2006;367:1241-1246.

Urol. 2005;174:353-359.

Int. 2004;94:1132-1135.

Artif Organs. 2008;32:91-99.

Urol. 2010;28:227-232.

302.

Biomaterials. 2009;30:3259-3270.

Biomed Mater Res A. 2010 ;94:712-719.

regeneration. World J Urol. 2008;26:323-326.


[19] Yao C, Prével P, Koch S, Schenck P, Noah EM, Pallua N, et al. Modification of collagen matrices for enhancing angiogenesis. Cells Tissues Organs. 2004;178:189-196. [20] Kundu AK, Gelman J, Tyson DR. Composite thin film and electrospun biomaterials for

[21] Baumert H, Simon P, Hekmati M, Fromont G, Levy M, Balaton A, et al. Development of

[22] Gu GL, Zhu YJ, Xia SJ, Zhang J, Jiang JT, Hong Y, et al.Peritoneal cavity as bioreactor to

[23] Farhat WA, Yeger H. Does mechanical stimulation have any role in urinary bladder

[24] A novel bioreactor to simulate urinary bladder mechanical properties and compliance

[25] Khang D, Lu J, Yao C, Haberstroh KM, Webster TJ.The role of nanometer and sub-

[26] Chun YW, Lim H, Webster TJ, Haberstroh KM. Nanostructured bladder tissue replacements.Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2010 Aug 20. [27] Mondalek FG, Lawrence BJ, Kropp BP, Grady BP, Fung KM, Madihally SV,et al. The

[28] Harrison BS, Eberli D, Lee SJ, Atala A, Yoo JJ. Oxygen producing biomaterials for tissue

[29] Oh SH, Ward CL, Atala A, Yoo JJ, Harrison BS. Oxygen generating scaffolds for enhancing engineered tissue survival. Biomaterials. 2009;30:757-762. [30] Kropp BP.Small-intestinal submucosa for bladder augmentation:a review of preclinical

[31] Kropp BP, Eppley BL, Prevel CD, Rippy MK, Harruff RC, Badylak SF, et al.

[32] Kropp BP, Sawyer BD, Shannon HE, Rippy MK, Badylak SF, Adams MC, et al.

[33] Kropp BP, Cheng EY, Lin HK, Zhang Y. Reliable and reproducible bladder regeneration using unseeded distal small intestinal submucosa. J Urol. 2004;172:1710-1713. [34] Zhang Y, Kropp BP, Moore P, Cowan R, Furness PD 3rd, Kolligian ME, et al. Coculture

[35] Liu Y, Bharadwaj S, Lee SJ, Atala A, Zhang Y. Optimization of a natural collagen

Experimental assessment of small intestinal submucosa as bladder wall substitute.

Characterization of small intestinal submucosa regenerated canine detrusor: assessment of reinnervation, in vitro compliance and contractility. J

of bladder urothelial and smooth muscle cells on small intestinal submucosa: potential application for tissue engineering technology. J Urol. 2000;164:928-934;

scaffold to aid cell–matrix penetration for urologic tissue engineering.

intestinal submucosa biomaterials. Biomaterials 2008,29:1159–1166

a seeded scaffold in the great omentum: feasibility of an in vivo bioreactor for

grow autologous tubular urethral grafts in a rabbit model. World J Urol. 2010;28:

micron surface features on vascular and bone cell adhesion on

incorporation of poly(lactic-co-glycolic) acid nanoparticles into porcine small

urologic tissue reconstruction. Biotechnol Bioeng. 2011;108:207-215.

bladder tissue engineering. Eur Urol. 2007;52:884-890.

tissue engineering. World J Urol. 2008;26:301-305.

for bladder functional tissue engineering

titanium.Biomaterials 2008, 29:970–983.

regeneration. Biomaterials. 2007;28:4628-4634.

studies. World J Urol. 1998;16:262-267.

Urology. 1995;46:396-400.

Urol. 1996;156:599-607.

discussion 934-935

Biomaterials. 2009;30:3865-3873.

227-232.


**20** 

Abir El-Sadik

*Kingdom of Saudi Arabia* 

**Novel Promises of Nanotechnology** 

*King Saud Bin Abdulaziz University for Health Sciences, Riyadh* 

The term 'nanotechnology' refers to technology that deals with structures and devices of nanometer (10 – 9 meter) size. It involves the design, fabrication and utilization of materials of nanoscale dimensions (Gao & Xu, 2009). The resulting nanomaterials exhibit chemical, physical and biological properties that can differ significantly from those of bulk material. These products can be categorized into metals, ceramics, polymers or composite materials that have nanoscale features. The limited size of their particles leads to a high surface area to volume ratio, improved solubility, multifunctionality, high electrical and heat conductivity and improved surface catalytic activity. (El-Sadik et al., 2010). All these phenomena allow give nanoparticles to interact with biological systems at cellular and molecular levels. These interactions enhance the biomedical applications of nanotechnology giving great promise for improving disease prevention, diagnosis, treatment and in particular tissue regeneration

Since natural human tissues include nano-scale subcellular and extracellular components, artificial nanomaterials mimic the scales of tissue components (Zhang & Webster, 2009). Cells make contact with other cells and with the extracellular matrix with membrnes that have nanoscale features. It has been shown that nanomaterials, with their biomimetic features, can accelerate the rate of cell growth and proliferation and promote tissue acceptance due to reduced immune response (Oh et al., 2009). One of the most useful properties of nanomaterials, which have been extensively investigated, is their ability to interact with proteins that control cell functions. This may make nanomaterials very useful, and perhaps even necessary, tools for regenerating various tissues such as those of the bone,

Although a series of technological improvements in tissue regeneration have been acheived using conventional methods, a variety of problems still faces current implants. Nanotechnology could provide several solutions to these problems. A wide range of nanomaterials have been made from organic and inorganic composites, just like conventional materials. However, nanotehnology has the ability to control material properties more closely by assembling components at the nanoscale. These nanomaterials (nanoparticles, nanotubes, nanofibers, nanoclusters, nanocrystals, nanowires, nanorods and

cartilage, blood vessels and nervous system (Liu & Webster, 2007).

**1. Introduction** 

(Murthy, 2007).

**for Tissue Regeneration** 

*Anatomy and Embryology, Basic Sciences Department,* 


## **Novel Promises of Nanotechnology for Tissue Regeneration**

#### Abir El-Sadik

*Anatomy and Embryology, Basic Sciences Department, King Saud Bin Abdulaziz University for Health Sciences, Riyadh Kingdom of Saudi Arabia* 

#### **1. Introduction**

452 Tissue Regeneration – From Basic Biology to Clinical Application

[52] Schlote N, Wefer J, Sievert KD. Acellular matrix for functional reconstruction of the

[53] Donkov II, Bashir A, Elenkov CH, Panchev PK. Dorsal onlay augmentation

[54] Palminteri E, Berdondini E, Colombo F, Austoni E. Small intestinal submucosa (SIS)

[55] Fiala R, Vidlar A, Vrtal R, Belej K, Student V. Porcine small intestinal submucosa graft for repair of anterior urethral strictures. Eur Urol. 2007;51:1702-1708; [56] Farahat YA, Elbahnasy AM, El-Gamal OM, Ramadan AR, El-Abd SA, Taha MR.

[57] Bhargava S, Patterson JM, Inman RD, MacNeil S, Chapple CR. Tissue-Engineered

[58] Raya-Rivera A, Esquiliano DR, Yoo JJ, Lopez-Bayghen E, Soker S, Atala A. Tissue-

strictures of the bulbar urethra. Int J Urol. 2006;13:1415-1417.

observational study. Lancet. 2011;377:1175-1182

graft urethroplasty: short-term results. Eur Urol. 2007;51:1695-1701

1212

1269.

urogenital tract. Special form of "tissue engineering"?. Urologe A. 2004;43:1209-

urethroplasty with small intestinal submucosa: modified Barbagli technique for

Endoscopic urethroplasty using small intestinal submucosal patch in cases of recurrent urethral stricture: a preliminary study. J Endourol. 2009;23:2001-2005

Buccal Mucosa Urethroplasty—Clinical Outcomes. Eur Urol. 2008;53:1263-

engineered autologous urethras for patients who need reconstruction: an

The term 'nanotechnology' refers to technology that deals with structures and devices of nanometer (10 – 9 meter) size. It involves the design, fabrication and utilization of materials of nanoscale dimensions (Gao & Xu, 2009). The resulting nanomaterials exhibit chemical, physical and biological properties that can differ significantly from those of bulk material. These products can be categorized into metals, ceramics, polymers or composite materials that have nanoscale features. The limited size of their particles leads to a high surface area to volume ratio, improved solubility, multifunctionality, high electrical and heat conductivity and improved surface catalytic activity. (El-Sadik et al., 2010). All these phenomena allow give nanoparticles to interact with biological systems at cellular and molecular levels. These interactions enhance the biomedical applications of nanotechnology giving great promise for improving disease prevention, diagnosis, treatment and in particular tissue regeneration (Murthy, 2007).

Since natural human tissues include nano-scale subcellular and extracellular components, artificial nanomaterials mimic the scales of tissue components (Zhang & Webster, 2009). Cells make contact with other cells and with the extracellular matrix with membrnes that have nanoscale features. It has been shown that nanomaterials, with their biomimetic features, can accelerate the rate of cell growth and proliferation and promote tissue acceptance due to reduced immune response (Oh et al., 2009). One of the most useful properties of nanomaterials, which have been extensively investigated, is their ability to interact with proteins that control cell functions. This may make nanomaterials very useful, and perhaps even necessary, tools for regenerating various tissues such as those of the bone, cartilage, blood vessels and nervous system (Liu & Webster, 2007).

Although a series of technological improvements in tissue regeneration have been acheived using conventional methods, a variety of problems still faces current implants. Nanotechnology could provide several solutions to these problems. A wide range of nanomaterials have been made from organic and inorganic composites, just like conventional materials. However, nanotehnology has the ability to control material properties more closely by assembling components at the nanoscale. These nanomaterials (nanoparticles, nanotubes, nanofibers, nanoclusters, nanocrystals, nanowires, nanorods and

Novel Promises of Nanotechnology for Tissue Regeneration 455

chondrogenic synovium (Huang et al., 2008 & Shi et al., 2009). Fibrin polylactide caprolactone nanoparticles have been designed to induce chondrogenic differentiation in mesenchymal stem cells. These complex nanoparticles facilitated the upregulation of chondrogenesis marker genes. In addition, they effectively sustained chondrogenic differentiation and enhanced chondral extracellular matrix deposition by human adipogenic stem cells. Fibrin polylactide caprolactone nanoparticle complexes could be effectively used for in situ cartilage tissue

The application of nanotechnology to stem cell biology might help to maximize therapeutic benefits and minimize possible undesired effects of stem cell therapy, through delivery of sufficient stem cells to the regions of interest with the smallest number of cells to untargeted regions. Tracking the fate, distribution, proliferation, differentiation of engulfed stem cells employed in tissue regeneration is essential to understand the mechanisms of participation of the cells in tissue repair. Nanotechnology can improve several techniques that would enable non-invasive detection of transplanted stem cells within the desired organs. Iron oxide nanoparticles are inorganic nanoparticles that can be synthesized easily in large quantities and different sizes using simple methods. Several studies reported that when iron oxide nanoparticles bind to the external cell membrane, they do not affect cell viability, although they may detach from the cell membrane or interfere with cell surface interactions (Bulte & Kraitchman, 2004). Superparamagnetic iron oxide nanoparticles are successfully internalized via endocytosis in human mesenchymal stem cells. After their uptake, they are located inside cytoplasmic vesicles. Then, they are transferred to lysosomes in which degradation of the nanoparticles occurs, releasing free iron into the cytoplasm (Jing et al., 2008). Coating the surface of iron oxide nanoparticles modifies the surface of the particles for efficient uptake with minimum side effects on the cells. Coating superparamagnetic iron oxide nanoparticles with dextran improves their stability and solubility and prevents their aggregation. Another example of coating the surface of nanoparticles is provided by coating the superparamagnetic iron oxide nanoparticles with gold. The gold provides an inert shell around the nanoparticles and protects them from rapid dissolution within cytoplasmic endosomes and enhances magnetic resonance imaging (MRI) contrast. It has been shown, however, that dissolved iron oxide nanoparticles may produce free hydroxyl radicals which increase the rate of apoptosis and alterations in cellular metabolism (Emerit et al., 2001).

Concerning the effects of iron oxide nanoparticles on stem cell behaviour, magnetite iron oxide cationic liposomes can be applied efficiently to mesenchymal stem cell techniques. Mesenchymal stem cells incubated in osteogenic medium with these nanoparticles changed their shape from fibroblastic to polygonal, formed calcium nodules and increased in number five-fold compared with controls (Ito et al., 2004). In addition, superparamagnetic iron oxide nanoparticles have been shown to enhance the survival rate of stem cells up to 99%, indicating that these nanoparticles improve stem cell viability (Delcroix et al., 2009). Moreover, superparamagnetic iron oxide nanoparticles did not influence the morphology, cell cycle, telomerase activity, proliferation or differentiation ability of labelled neural stem cells (Kea et al., 2009). Superparamagnetic iron oxide nanoparticles have been successfully applied to tracking the fate of several types of stem cells. For example, the migration of embryonic stem cells and bone marrow mesenchymal stem cells labelled with iron oxide nanoparticles towards a lesion site has been tracked using MRI. This labelling technique offers high resolution, speed, easy access and 3-dimensional capabilities and provides

regeneration from human stem cells (Jung et al., 2009).

nanofilms) can be fabricated by multiple and available nanotechnologies. Electrospining, self assembly, phase separation, photolithography, thin film deposition, chemical etching, chemical vapor deposition and electron beam lithography are all techniques currently used to synthesize nanomaterials with ordered or random nanotopographies (Chen & Ma, 2004).

Conventional tissue replacement, using allografts and autografts, cannot satisfy high performance demands and improvements are necessary. Nanotechnology has been used to fabricate cytocompatible biomimetic nanomaterials that provide biological substitutes useful in restoring and improving tissue functions. Moreover, 2-dimensional tissue cell culture systems on flat glass, coated petri dishes or plastic substrates cannot simulate the natural tissue microenvironments. Normal tissue cells are located in a complex network of 3 dimensional extracellular matrix with nanoscale fibers. Nanomaterials could be fabricated that accurately simulate the dimensions and architecture of natural human tissue, allowing significantly improved performance of the cultured cells (Gelain et al., 2006). The composition and topography of a tissue engineered material could even produce cellenvironment interactions that determine the implant fate. Nanomaterials need to be designed to be biocompatible and to function without interrupting other physiological processes. In principle, they can promote normal cell growth and differentiation without any adverse tissue reaction. These nanomaterials must be biodegradable either to be removed via degradation or absorption to leave only native tissue. In addition, nanomaterials used in tissue regeneration should possess biomimetic features that allow cells to react normally to internal and external stimuli and to exchange the signals between those cells and the external environment.

This chapter reviews recent progress in the synthesis of nanomaterials for improving stem cell behavior and tissue regeneration. In addition, it highlights potentially valuable applications of nanotechnology in specific tissue regeneration.

#### **2. Effects of nanomaterials on stem cell behaviour and development of tissue regeneration**

Nanotechnology is an extremely promising advancement in synthetic methodologies used to functionalize nanomaterials with biomolecules. Nanomolecules could be modified to desired sizes, shapes, compositions and properties producing different types applied in tissue regeneration such as nanoparticles, nanosurfaces and nanoscaffolds.

#### **2.1 Nanoparticles**

Several studies have investigated the influences of different types of nanoparticles on the behaviour of stem cells applied in tissue regeneration. The effects of mesoporous silica nanoparticles conjugated with fluorescein isothiocyanate on human bone marrow mesenchymal stem cells has been investigated by several researchers. Internalization of silica nanoparticles into stem cells is mediated by both clathrin and actin-dependent endocytosis. Once inside the cell, the nanoparticles escaped the endolysosomal vesicles and did not affect stem cell viability or proliferation. They enhanced actin polymerization in mesenchymal stem cells. Moreover, regular osteogenic differentiation was successfully induced in the mesenchymal stem cells after the uptake of mesoporous silica nanoparticles in highly

nanofilms) can be fabricated by multiple and available nanotechnologies. Electrospining, self assembly, phase separation, photolithography, thin film deposition, chemical etching, chemical vapor deposition and electron beam lithography are all techniques currently used to synthesize nanomaterials with ordered or random nanotopographies (Chen & Ma, 2004). Conventional tissue replacement, using allografts and autografts, cannot satisfy high performance demands and improvements are necessary. Nanotechnology has been used to fabricate cytocompatible biomimetic nanomaterials that provide biological substitutes useful in restoring and improving tissue functions. Moreover, 2-dimensional tissue cell culture systems on flat glass, coated petri dishes or plastic substrates cannot simulate the natural tissue microenvironments. Normal tissue cells are located in a complex network of 3 dimensional extracellular matrix with nanoscale fibers. Nanomaterials could be fabricated that accurately simulate the dimensions and architecture of natural human tissue, allowing significantly improved performance of the cultured cells (Gelain et al., 2006). The composition and topography of a tissue engineered material could even produce cellenvironment interactions that determine the implant fate. Nanomaterials need to be designed to be biocompatible and to function without interrupting other physiological processes. In principle, they can promote normal cell growth and differentiation without any adverse tissue reaction. These nanomaterials must be biodegradable either to be removed via degradation or absorption to leave only native tissue. In addition, nanomaterials used in tissue regeneration should possess biomimetic features that allow cells to react normally to internal and external stimuli and to exchange the signals between

This chapter reviews recent progress in the synthesis of nanomaterials for improving stem cell behavior and tissue regeneration. In addition, it highlights potentially valuable

**2. Effects of nanomaterials on stem cell behaviour and development of tissue** 

Nanotechnology is an extremely promising advancement in synthetic methodologies used to functionalize nanomaterials with biomolecules. Nanomolecules could be modified to desired sizes, shapes, compositions and properties producing different types applied in

Several studies have investigated the influences of different types of nanoparticles on the behaviour of stem cells applied in tissue regeneration. The effects of mesoporous silica nanoparticles conjugated with fluorescein isothiocyanate on human bone marrow mesenchymal stem cells has been investigated by several researchers. Internalization of silica nanoparticles into stem cells is mediated by both clathrin and actin-dependent endocytosis. Once inside the cell, the nanoparticles escaped the endolysosomal vesicles and did not affect stem cell viability or proliferation. They enhanced actin polymerization in mesenchymal stem cells. Moreover, regular osteogenic differentiation was successfully induced in the mesenchymal stem cells after the uptake of mesoporous silica nanoparticles in highly

those cells and the external environment.

**regeneration** 

**2.1 Nanoparticles** 

applications of nanotechnology in specific tissue regeneration.

tissue regeneration such as nanoparticles, nanosurfaces and nanoscaffolds.

chondrogenic synovium (Huang et al., 2008 & Shi et al., 2009). Fibrin polylactide caprolactone nanoparticles have been designed to induce chondrogenic differentiation in mesenchymal stem cells. These complex nanoparticles facilitated the upregulation of chondrogenesis marker genes. In addition, they effectively sustained chondrogenic differentiation and enhanced chondral extracellular matrix deposition by human adipogenic stem cells. Fibrin polylactide caprolactone nanoparticle complexes could be effectively used for in situ cartilage tissue regeneration from human stem cells (Jung et al., 2009).

The application of nanotechnology to stem cell biology might help to maximize therapeutic benefits and minimize possible undesired effects of stem cell therapy, through delivery of sufficient stem cells to the regions of interest with the smallest number of cells to untargeted regions. Tracking the fate, distribution, proliferation, differentiation of engulfed stem cells employed in tissue regeneration is essential to understand the mechanisms of participation of the cells in tissue repair. Nanotechnology can improve several techniques that would enable non-invasive detection of transplanted stem cells within the desired organs. Iron oxide nanoparticles are inorganic nanoparticles that can be synthesized easily in large quantities and different sizes using simple methods. Several studies reported that when iron oxide nanoparticles bind to the external cell membrane, they do not affect cell viability, although they may detach from the cell membrane or interfere with cell surface interactions (Bulte & Kraitchman, 2004). Superparamagnetic iron oxide nanoparticles are successfully internalized via endocytosis in human mesenchymal stem cells. After their uptake, they are located inside cytoplasmic vesicles. Then, they are transferred to lysosomes in which degradation of the nanoparticles occurs, releasing free iron into the cytoplasm (Jing et al., 2008). Coating the surface of iron oxide nanoparticles modifies the surface of the particles for efficient uptake with minimum side effects on the cells. Coating superparamagnetic iron oxide nanoparticles with dextran improves their stability and solubility and prevents their aggregation. Another example of coating the surface of nanoparticles is provided by coating the superparamagnetic iron oxide nanoparticles with gold. The gold provides an inert shell around the nanoparticles and protects them from rapid dissolution within cytoplasmic endosomes and enhances magnetic resonance imaging (MRI) contrast. It has been shown, however, that dissolved iron oxide nanoparticles may produce free hydroxyl radicals which increase the rate of apoptosis and alterations in cellular metabolism (Emerit et al., 2001).

Concerning the effects of iron oxide nanoparticles on stem cell behaviour, magnetite iron oxide cationic liposomes can be applied efficiently to mesenchymal stem cell techniques. Mesenchymal stem cells incubated in osteogenic medium with these nanoparticles changed their shape from fibroblastic to polygonal, formed calcium nodules and increased in number five-fold compared with controls (Ito et al., 2004). In addition, superparamagnetic iron oxide nanoparticles have been shown to enhance the survival rate of stem cells up to 99%, indicating that these nanoparticles improve stem cell viability (Delcroix et al., 2009). Moreover, superparamagnetic iron oxide nanoparticles did not influence the morphology, cell cycle, telomerase activity, proliferation or differentiation ability of labelled neural stem cells (Kea et al., 2009). Superparamagnetic iron oxide nanoparticles have been successfully applied to tracking the fate of several types of stem cells. For example, the migration of embryonic stem cells and bone marrow mesenchymal stem cells labelled with iron oxide nanoparticles towards a lesion site has been tracked using MRI. This labelling technique offers high resolution, speed, easy access and 3-dimensional capabilities and provides

Novel Promises of Nanotechnology for Tissue Regeneration 457

the nanotopography of their environment, which influences their cytoskeletal organization, attachment, and migration. Nanofabrication techniques provide several types of

Nanosurfaces of different materials with structural modification, such as the presence of large, medium and small nanoscale grooves, pores, pits, ridges and nodules can be recognized by cultured cells. A wide range of cell types, such as fibroblasts (Dalby et al., 2003b), osteoblasts (Lenhert et al., 2005) and mesenchymal stem cells (Biggs et al., 2008), are influenced by nanoscale grooves with dimensions that mimic those in vivo. Cellular morphology depends on cell type and on groove depth and width. Mesenchymal stem cells seeded on nanogrooves respond by aligning their shape and elongation in the direction of the grooves (Dalby et al., 2003b). Human osteoblasts cultured on ordered nanoscale groove/ridge arrays, fabricated by photolithography, were affected significantly (Biggs et al., 2008). The authors seeded human osteoblasts on grooves of 330 nm depth and different widths (10, 25 and 100 µm in width). They concluded that adhesion formation was not affected in 100 µm wide groove/ridge arrays, although upregulation of genes involved in skeletal development was induced. In addition, increased osteospecific functions were observed. 25 µm wide grooves/ ridges were shown to be associated with a reduction in supermature adhesions and an increase in focal complex formation. However, that osteoblast adhesion was significantly reduced in 10 µm wide groove/ridge arrays. Moreover, grooves manufactured on nanosurfaces promoted the elongation and nuclear polarization in the cultured cells (Charest et al., 2004). Cell membranes stopped at the largest grooves but bridged over the narrowest and deepest ones (Matsuzaka et al., 2003). Electron beam lithography has also been used to generate nanoscale patterns for culturing mesenchymal stem cells (Dalby, 2009). The patterns ranged from highly ordered through controlled disorder, to total randomness. The authors concluded that nanoscale change in surface topography altered mesenchymal stem cell differentiation. Successful osteoconversion of the cultured cells using ± 50 nm level of disorder was demonstrated. The cells focal adhesions interacted with the material surface and affected by several signalling pathways, such as G protein and cytoskeletal signalling. These signalling factors modulated cell sensing, morphology, contractility, proliferation and differentiation. Altering the nanotopography of the surface material influenced the cytoskeletal arrangements (Curtis et al., 2006). Mechanical changes were transmitted from the cytoskeleton to the nucleus,

affecting the genomic expression patterns and cell phenotype (Dalby et al., 2007).

Among hard carbon coatings, nanocrystalline diamond has been applied successfully to cultured osteogenic and endothelial cells. Nanocrystalline diamond possesses promising electrical and optical properties, high hardness, low friction coefficient and good compatibility (Bacakova et al., 2007). Nanocrystalline diamond has been used in the form of films to improve the mechanical and physical properties of body implants. In addition, it has been shown to attract cell colonization, its surface nanostructure simulating the architecture of extracellular matrix molecules. Nanocrystalline diamond layers deposited on silicon substrates improves the adhesion and growth of osteogenic and endothelial cells (Grausova et al., 2008). The authors concluded that these nanostructured surfaces gave good support for cellular viability and proliferation and could be applied usefully in tissue regeneration. Furthermore, ultrasmooth nanostructured diamond has been used in

nanosurfaces for tissue regeneration.

information not only for the transplanted cells, but also for the surrounding tissues, reporting edema or inflammation that may affect the fate of the grafted cells and reduce the recovery of damaged tissue (Sykova & Jendelova, 2007). Another example is tracking human mesenchymal stem cells labelled with superparamagnetic iron oxide nanoparticles after transplantation for articular cartilage repair using MRI (Au et al., 2009). These observations have demonstrated the ability of using iron oxide nanoparticles to be useful in monitoring and tracking the fate of transplanted stem cells apparently without affecting their behaviour, although, selection of the type and concentration of nanoparticles is critically important.

In addition to iron oxide nanoparticles, quantum dots have been much used for cell tracking in in tissue regeneration. Quantum dots are fluorescent semiconducting nanocrystals that overcome the limitations of conventional labelling methods. Several researchers have studied the application of quantum dots to monitoring physiological changes inside living cells by labelling the intracellular organelles or specific proteins with quantum dots. They could monitor cellular migration, track cell lineage and investigate stem cell behaviour. Quantum dots can bind to individual molecules on the cell surface and serve in tracking the motion of those molecules. For example, quantum dots have been applied to demonstrate changes in integrin dynamics during osteogenic differentiation of human bone marrow cells (Chen et al., 2007). Numerous studies have demonstrated a variety of techniques of the cellular uptake of quantum dots. These nanoparticles can be delivered into cells by microinjection, endocytosis, liposome-mediated transfection and special peptide delivery (Chang et al., 2008). Delivered quantum dots were found to escaping lysosomal degradation at the beginning of the uptake. Thereafter, lysosome expression was enhanced and all cellular quantum dots were shown in lysosome vesicles. After the uptake of quantum dots, into several types of stem cells, such as mesenchymal stem cells, cytoskeletal reorganization took place. This action revealed the formation of wide and flat leading lamellipodia filled with a dense actin network (Chang et al., 2009). Human mesenchymal stem cells labelled with quantum dots represented the same viability comparing with the unlabelled human mesenchymal stem cells from the same subpopulation (Shah et al., 2007), suggesting that quantum dots could be used safely for long term labelling of stem cells. Moreover, embryonic stem cells could be labelled with quantum dots for cellular tracking in vivo without affecting the viability, proliferation or differentiation of the embryonic stem cells (Lin et al., 2007). These studies demonstrated that quantum dots could enable cellular and molecular imaging and tracking the fate of stem and progenitor cells used in tissue regeneration with high sensitivity and high spatial resolution. These applications are supported by an extensive number of advanced imaging techniques, giving a great impact on tissue regeneration studies.

#### **2.2 Nanosurfaces**

Mammalian cells are surrounded by nanostructures formed by biomolecules arranged geometrically in different configurations. These arrangements affect cell behaviour by producing chemical signals such as growth factors or physical signals such as tensile forces caused by interactions with the surrounding nanostructured extracellular matrix. Nanotechnology provides nanotopographical surfaces that can guide cellular adhesion, spreading, morphology, proliferation and differentiation. Cells react differently according to

information not only for the transplanted cells, but also for the surrounding tissues, reporting edema or inflammation that may affect the fate of the grafted cells and reduce the recovery of damaged tissue (Sykova & Jendelova, 2007). Another example is tracking human mesenchymal stem cells labelled with superparamagnetic iron oxide nanoparticles after transplantation for articular cartilage repair using MRI (Au et al., 2009). These observations have demonstrated the ability of using iron oxide nanoparticles to be useful in monitoring and tracking the fate of transplanted stem cells apparently without affecting their behaviour, although, selection of the type and concentration of nanoparticles is

In addition to iron oxide nanoparticles, quantum dots have been much used for cell tracking in in tissue regeneration. Quantum dots are fluorescent semiconducting nanocrystals that overcome the limitations of conventional labelling methods. Several researchers have studied the application of quantum dots to monitoring physiological changes inside living cells by labelling the intracellular organelles or specific proteins with quantum dots. They could monitor cellular migration, track cell lineage and investigate stem cell behaviour. Quantum dots can bind to individual molecules on the cell surface and serve in tracking the motion of those molecules. For example, quantum dots have been applied to demonstrate changes in integrin dynamics during osteogenic differentiation of human bone marrow cells (Chen et al., 2007). Numerous studies have demonstrated a variety of techniques of the cellular uptake of quantum dots. These nanoparticles can be delivered into cells by microinjection, endocytosis, liposome-mediated transfection and special peptide delivery (Chang et al., 2008). Delivered quantum dots were found to escaping lysosomal degradation at the beginning of the uptake. Thereafter, lysosome expression was enhanced and all cellular quantum dots were shown in lysosome vesicles. After the uptake of quantum dots, into several types of stem cells, such as mesenchymal stem cells, cytoskeletal reorganization took place. This action revealed the formation of wide and flat leading lamellipodia filled with a dense actin network (Chang et al., 2009). Human mesenchymal stem cells labelled with quantum dots represented the same viability comparing with the unlabelled human mesenchymal stem cells from the same subpopulation (Shah et al., 2007), suggesting that quantum dots could be used safely for long term labelling of stem cells. Moreover, embryonic stem cells could be labelled with quantum dots for cellular tracking in vivo without affecting the viability, proliferation or differentiation of the embryonic stem cells (Lin et al., 2007). These studies demonstrated that quantum dots could enable cellular and molecular imaging and tracking the fate of stem and progenitor cells used in tissue regeneration with high sensitivity and high spatial resolution. These applications are supported by an extensive number of advanced imaging techniques, giving a great impact

Mammalian cells are surrounded by nanostructures formed by biomolecules arranged geometrically in different configurations. These arrangements affect cell behaviour by producing chemical signals such as growth factors or physical signals such as tensile forces caused by interactions with the surrounding nanostructured extracellular matrix. Nanotechnology provides nanotopographical surfaces that can guide cellular adhesion, spreading, morphology, proliferation and differentiation. Cells react differently according to

critically important.

on tissue regeneration studies.

**2.2 Nanosurfaces** 

the nanotopography of their environment, which influences their cytoskeletal organization, attachment, and migration. Nanofabrication techniques provide several types of nanosurfaces for tissue regeneration.

Nanosurfaces of different materials with structural modification, such as the presence of large, medium and small nanoscale grooves, pores, pits, ridges and nodules can be recognized by cultured cells. A wide range of cell types, such as fibroblasts (Dalby et al., 2003b), osteoblasts (Lenhert et al., 2005) and mesenchymal stem cells (Biggs et al., 2008), are influenced by nanoscale grooves with dimensions that mimic those in vivo. Cellular morphology depends on cell type and on groove depth and width. Mesenchymal stem cells seeded on nanogrooves respond by aligning their shape and elongation in the direction of the grooves (Dalby et al., 2003b). Human osteoblasts cultured on ordered nanoscale groove/ridge arrays, fabricated by photolithography, were affected significantly (Biggs et al., 2008). The authors seeded human osteoblasts on grooves of 330 nm depth and different widths (10, 25 and 100 µm in width). They concluded that adhesion formation was not affected in 100 µm wide groove/ridge arrays, although upregulation of genes involved in skeletal development was induced. In addition, increased osteospecific functions were observed. 25 µm wide grooves/ ridges were shown to be associated with a reduction in supermature adhesions and an increase in focal complex formation. However, that osteoblast adhesion was significantly reduced in 10 µm wide groove/ridge arrays. Moreover, grooves manufactured on nanosurfaces promoted the elongation and nuclear polarization in the cultured cells (Charest et al., 2004). Cell membranes stopped at the largest grooves but bridged over the narrowest and deepest ones (Matsuzaka et al., 2003). Electron beam lithography has also been used to generate nanoscale patterns for culturing mesenchymal stem cells (Dalby, 2009). The patterns ranged from highly ordered through controlled disorder, to total randomness. The authors concluded that nanoscale change in surface topography altered mesenchymal stem cell differentiation. Successful osteoconversion of the cultured cells using ± 50 nm level of disorder was demonstrated. The cells focal adhesions interacted with the material surface and affected by several signalling pathways, such as G protein and cytoskeletal signalling. These signalling factors modulated cell sensing, morphology, contractility, proliferation and differentiation. Altering the nanotopography of the surface material influenced the cytoskeletal arrangements (Curtis et al., 2006). Mechanical changes were transmitted from the cytoskeleton to the nucleus, affecting the genomic expression patterns and cell phenotype (Dalby et al., 2007).

Among hard carbon coatings, nanocrystalline diamond has been applied successfully to cultured osteogenic and endothelial cells. Nanocrystalline diamond possesses promising electrical and optical properties, high hardness, low friction coefficient and good compatibility (Bacakova et al., 2007). Nanocrystalline diamond has been used in the form of films to improve the mechanical and physical properties of body implants. In addition, it has been shown to attract cell colonization, its surface nanostructure simulating the architecture of extracellular matrix molecules. Nanocrystalline diamond layers deposited on silicon substrates improves the adhesion and growth of osteogenic and endothelial cells (Grausova et al., 2008). The authors concluded that these nanostructured surfaces gave good support for cellular viability and proliferation and could be applied usefully in tissue regeneration. Furthermore, ultrasmooth nanostructured diamond has been used in

Novel Promises of Nanotechnology for Tissue Regeneration 459

surface roughness. Higher numbers of attached cells were observed on 4-layer titanium dioxide thin film than on a 1-layer thin film, with a faster rate of spreading on the rougher surface (Kommireddy et al., 2006). Moreover, multilayered and functionalized titanium films composed of chitosan and plasmid DNA demonstrated significant high transfection efficiency in mesenchymal stem cells (Hu et al., 2009). The authors reported high production levels of alkaline phosphatase and osteocalcin. They concluded that multilayered titanium films with chitosan and plasmid DNA promoted the differentiation of osteoprogenitor cells

Nanotechnology provides the tissue regeneration field with nanostructures that might accurately simulate the natural 3-dimensional microenvironment of cells. This approach provides a complex network of nanoscale fibers and extracellular ligands, such as many types of collagens, laminin and fibronectin, that are poorly reproduced in the conventional 2-dimensional systems. Growth of cells in 2-dimensional cultures has been shown to reduce the production of particular extracellular matrix proteins, with consequent morphological changes and increase in spreading. The advancement in the technology of nanostructures enhances the scope of fabricating 3-dimensional nanoscaffolds that could potentially mimic the architecture of natural human tissue. These nanostructured scaffolds could control and direct cellular behaviour and interactions with the extracellular matrix. Scaffolds have been designed in the form of nanofibers, nanotubes, nanowires, nanorods, nanocrystals and nanofilms. These nanostructured scaffolds with their biomimetic features and excellent physicochemical properties, stimulated cellular adhesion, growth, morphology, proliferation, altered gene expression and promoted cellular differentiation. The structural features of these nanoscaffolds were engineered according to the nature of cell response which was desired. The scaffolds were designed in a manner that provided a surface to promote cell attachment, spreading and growth while encouraging the formation of a porous network that offered a suitable path for nutrient transmission and tissue ingrowth (Chen & Ma, 2004). These novel nanoscaffolds had excellent mechanical properties that offered structural support until the new tissue would be formed, as they degraded at a rate matching the new tissue formation and provided substrate for cell migration and survival. They were biocompatible and the products of their degradation were also biocompatible (Smith et al., 2010). These nanostructured scaffolds provided the functional role of the native extracellular matrix with growth factors that regulated the cell fate and bioactive peptide sequences that could bind receptors and activate intracellular signalling pathways

Several techniques have been designed for the fabrication of nanofibrous scaffolds to be employed in tissue regeneration. Electrospinning techniques have been the most commonly used. An electric field is applied to draw a polymer solution from an orifice to a collector, producing polymer fibers with diameters ranging in size from 50 nm to several microns. These resulted lengths mimicked that of native collagen fibrils (Baker et al., 2009). Several types of synthetic and natural biomaterials have been used to form nanofibrous scaffolds such as poly (caprolactone) (PCL), poly (lactic-co-glycolic acid) (PLGA) poly (L-lactic acid) (PLLA), collagen, gelatine and fibrinogen; molecules that have been applied extensively in

into mature osteoblasts over long time.

**2.3 Nanoscaffolds** 

(Boudreau & Jones, 1999).

orthopaedic implants. Several studies were performed on this material, the authors describing their surface modification techniques and cytocompatability (Clem et al., 2008). The studies demonstrated that hydrogen-terminated ultrasmooth nanostructured diamond surfaces supported robust mesenchymal stem cell adhesion and survival. However oxygen and fluorine terminated surfaces resisted cell adhesion. It was concluded that chemical and physical modifications of ultrasmooth nanostructured diamond could promote or prevent cell/biomaterial interactions. Moreover, mesenchymal stem cell adhesion and proliferation were significantly improved on ultrasmooth nanostructured diamond compared with the commonly used and biocompatible cobalt-chrome. There was also osteoblastic differentiation and deposition of mineralized matrix in mesenchymal stem cells. Ultrasmooth nanostructured diamond was found to reduce debris particle release from orthopaedic implants without influencing osseointegration.

Controllable self-assembly of nanonodules has been demonstrated to occur during chemical depositioning of materials on specifically conditioned microtopographical surfaces (Ogawa et al., 2008). The substrate could be a nonmetalic material such a as biodegradable polymer. The biological potential of the nanonodular surfaces affecting the behaviour of cultured cells using titanium dioxide has been investigated (Kubo et al., 2009). Titanium as a substrate material was proven to be non cytotoxic and was applied in therapeutic and implantable devices used in tissue regeneration. Micro-nano-hybrid surfaces, consisting of nanoscale nodules within microscale pits, were created by applying nanonodular self assembly techniques. These surfaces mimicked the biomineralized matrices with greater surface area and roughness. Changing the assembly time controlled the size of the nanonodules. The addition of nanonodules of different sizes (100 – 300 – 500 nm) to micropits selectively promoted osteoblast functions. In addition, these nanonodular topographies enhanced osteoblastic proliferation and differentiation. These advantages were 3 times greater in the nanonodules with a diameter of 300 nm within the micropits, when implanted in a rat femur model. Cell spread was enhanced on the micro- nano-hybrid surfaces. After 3 hours incubation, osteoblasts were shown to be larger and their cell processes and cytoskeletons started to develop on the nanonodular surfaces, while they remained small and circular on the micropit surface alone. Meanwhile, marked cytoplasmic localization of the focal adhesion protein vinculin was shown on the micro-nano-hybrid surfaces, compared with those on the micropit surface which had faint expression.

Another application of titanium in tissue regeneration is the use of nanocrystalline titanium surfaces. This type of nanometer surface roughness promotes osteoblasted adhesion. This nanosurface enhances cell growth and demonstrates extensive wear resistance due to high hardness and strength (Wang & Li, 2003). Cell compatability studies on nanosized titanium particles showed enhanced osteoblast function and largeer deposition of calcium minerals (Webster et al., 2000). One of the most effective nanostructured titanium surfaces for enhancing the attachment, proliferation and spreading of mesenchymal stem cells is layerby-layer assembled titanium dioxide nanoparticle thin films. This technique depends on electrostatic attraction between oppositely charged species such as titanium dioxide nanoparticles. The advantage of layer-by-layer assembly is that the adsorption of material cn be controlled with nanometer precision. Titanium dioxide thin films have been proved to be an optimal surface for rapid attachment and spreading of cells (Kommireddy et al., 2005). Increasing the number of layers in titanium dioxide thin films has been shown to increase surface roughness. Higher numbers of attached cells were observed on 4-layer titanium dioxide thin film than on a 1-layer thin film, with a faster rate of spreading on the rougher surface (Kommireddy et al., 2006). Moreover, multilayered and functionalized titanium films composed of chitosan and plasmid DNA demonstrated significant high transfection efficiency in mesenchymal stem cells (Hu et al., 2009). The authors reported high production levels of alkaline phosphatase and osteocalcin. They concluded that multilayered titanium films with chitosan and plasmid DNA promoted the differentiation of osteoprogenitor cells into mature osteoblasts over long time.

#### **2.3 Nanoscaffolds**

458 Tissue Regeneration – From Basic Biology to Clinical Application

orthopaedic implants. Several studies were performed on this material, the authors describing their surface modification techniques and cytocompatability (Clem et al., 2008). The studies demonstrated that hydrogen-terminated ultrasmooth nanostructured diamond surfaces supported robust mesenchymal stem cell adhesion and survival. However oxygen and fluorine terminated surfaces resisted cell adhesion. It was concluded that chemical and physical modifications of ultrasmooth nanostructured diamond could promote or prevent cell/biomaterial interactions. Moreover, mesenchymal stem cell adhesion and proliferation were significantly improved on ultrasmooth nanostructured diamond compared with the commonly used and biocompatible cobalt-chrome. There was also osteoblastic differentiation and deposition of mineralized matrix in mesenchymal stem cells. Ultrasmooth nanostructured diamond was found to reduce debris particle release from

Controllable self-assembly of nanonodules has been demonstrated to occur during chemical depositioning of materials on specifically conditioned microtopographical surfaces (Ogawa et al., 2008). The substrate could be a nonmetalic material such a as biodegradable polymer. The biological potential of the nanonodular surfaces affecting the behaviour of cultured cells using titanium dioxide has been investigated (Kubo et al., 2009). Titanium as a substrate material was proven to be non cytotoxic and was applied in therapeutic and implantable devices used in tissue regeneration. Micro-nano-hybrid surfaces, consisting of nanoscale nodules within microscale pits, were created by applying nanonodular self assembly techniques. These surfaces mimicked the biomineralized matrices with greater surface area and roughness. Changing the assembly time controlled the size of the nanonodules. The addition of nanonodules of different sizes (100 – 300 – 500 nm) to micropits selectively promoted osteoblast functions. In addition, these nanonodular topographies enhanced osteoblastic proliferation and differentiation. These advantages were 3 times greater in the nanonodules with a diameter of 300 nm within the micropits, when implanted in a rat femur model. Cell spread was enhanced on the micro- nano-hybrid surfaces. After 3 hours incubation, osteoblasts were shown to be larger and their cell processes and cytoskeletons started to develop on the nanonodular surfaces, while they remained small and circular on the micropit surface alone. Meanwhile, marked cytoplasmic localization of the focal adhesion protein vinculin was shown on the micro-nano-hybrid surfaces, compared with

Another application of titanium in tissue regeneration is the use of nanocrystalline titanium surfaces. This type of nanometer surface roughness promotes osteoblasted adhesion. This nanosurface enhances cell growth and demonstrates extensive wear resistance due to high hardness and strength (Wang & Li, 2003). Cell compatability studies on nanosized titanium particles showed enhanced osteoblast function and largeer deposition of calcium minerals (Webster et al., 2000). One of the most effective nanostructured titanium surfaces for enhancing the attachment, proliferation and spreading of mesenchymal stem cells is layerby-layer assembled titanium dioxide nanoparticle thin films. This technique depends on electrostatic attraction between oppositely charged species such as titanium dioxide nanoparticles. The advantage of layer-by-layer assembly is that the adsorption of material cn be controlled with nanometer precision. Titanium dioxide thin films have been proved to be an optimal surface for rapid attachment and spreading of cells (Kommireddy et al., 2005). Increasing the number of layers in titanium dioxide thin films has been shown to increase

orthopaedic implants without influencing osseointegration.

those on the micropit surface which had faint expression.

Nanotechnology provides the tissue regeneration field with nanostructures that might accurately simulate the natural 3-dimensional microenvironment of cells. This approach provides a complex network of nanoscale fibers and extracellular ligands, such as many types of collagens, laminin and fibronectin, that are poorly reproduced in the conventional 2-dimensional systems. Growth of cells in 2-dimensional cultures has been shown to reduce the production of particular extracellular matrix proteins, with consequent morphological changes and increase in spreading. The advancement in the technology of nanostructures enhances the scope of fabricating 3-dimensional nanoscaffolds that could potentially mimic the architecture of natural human tissue. These nanostructured scaffolds could control and direct cellular behaviour and interactions with the extracellular matrix. Scaffolds have been designed in the form of nanofibers, nanotubes, nanowires, nanorods, nanocrystals and nanofilms. These nanostructured scaffolds with their biomimetic features and excellent physicochemical properties, stimulated cellular adhesion, growth, morphology, proliferation, altered gene expression and promoted cellular differentiation. The structural features of these nanoscaffolds were engineered according to the nature of cell response which was desired. The scaffolds were designed in a manner that provided a surface to promote cell attachment, spreading and growth while encouraging the formation of a porous network that offered a suitable path for nutrient transmission and tissue ingrowth (Chen & Ma, 2004). These novel nanoscaffolds had excellent mechanical properties that offered structural support until the new tissue would be formed, as they degraded at a rate matching the new tissue formation and provided substrate for cell migration and survival. They were biocompatible and the products of their degradation were also biocompatible (Smith et al., 2010). These nanostructured scaffolds provided the functional role of the native extracellular matrix with growth factors that regulated the cell fate and bioactive peptide sequences that could bind receptors and activate intracellular signalling pathways (Boudreau & Jones, 1999).

Several techniques have been designed for the fabrication of nanofibrous scaffolds to be employed in tissue regeneration. Electrospinning techniques have been the most commonly used. An electric field is applied to draw a polymer solution from an orifice to a collector, producing polymer fibers with diameters ranging in size from 50 nm to several microns. These resulted lengths mimicked that of native collagen fibrils (Baker et al., 2009). Several types of synthetic and natural biomaterials have been used to form nanofibrous scaffolds such as poly (caprolactone) (PCL), poly (lactic-co-glycolic acid) (PLGA) poly (L-lactic acid) (PLLA), collagen, gelatine and fibrinogen; molecules that have been applied extensively in

Novel Promises of Nanotechnology for Tissue Regeneration 461

fabricated cytocompatible biomimetic nanomaterial scaffolds encapsulating cells, such as stem cells, chondrocytes and osteoblasts. In addition, to the dimensional similarity to bone/cartilage tissue, nanomaterials also exhibited unique surface properties, such as surface topography, surface chemistry, surface wettabilty and surface energy, due to their significantly increased surface area and roughness compared to conventional or micron structured materials. As is known, material surface properties mediate specific protein adsorption and bioactivity, such as fibronectin, vitronectin and laminin, before cells adhere on implants, further, they regulate cell behaviour and dictate tissue regeneration. Furthermore, an important criterion for designing orthopaedic implant materials is the formation of sufficient osseointegration between synthetic materials and bone tissue. Studies have demonstrated that nanostructured materials with cell-favourable surface properties could promote greater amounts of specific protein interactions to more efficiently stimulate new bone growth compared to conventional materials (Webster et al., 2001). This is one of the underlying reasons that nanomaterials are superior to conventional materials for bone growth. Therefore, by controlling surface properties, various nanophase ceramic, polymer, metal and composite scaffolds have been designed for bone/cartilage tissue engineering

There have been significant advances in the development of bone scaffolds with various compositions and 3 dimensional configurations using a variety of techniques such as the electrospining process for the fabrication of nanofibrous matrices. Several studies have reported the performance of nanofibrous materials in guiding cells to initially adhere to, and spread over, the nanostructures, as well as triggering them to secrete appropriate extracellular matrix molecules targeted to the bone and cartilage tissues. The boneassociated cells and the progenitor/stem cells showed initial responses which were anchorage-dependant. The nanofibrous substratum provided favourable conditions for cell anchorage and growth. Further osteoblastic differentiation and mineralization have also been reported to be regulated in a positive manner on nanofibrous surfaces (Woo et al., 2007). One particular requirement of bone tissue regeneration was that the scaffold should be porous, to incorporate large number of cells. The 3-dimensional scaffolds provided the necessary support for bone cells to attach, grow and differentiate and defined the overall shape of a bone tissue cultured transplant (Jang et al., 2009). Nanofibrous and nanotubular scaffolds were fabricated to mimic collagen fibers in bone and cartilage. Natural collagen is a triple helix self assembled into nanofibers of 300 nm in length and 1.5 nm in diameter. A new nanofiber composite was designed with the same self-assembly pattern as collagen and hydroxyapatite crystals in bone by directly nucleating and aligning the hydroxyapatite on the long axis of a nanofiber. Mesenchymal stem cell behaviour on self-assembled peptide amphiphile nanofiber scaffolds was investigated. Significantly enhanced osteogenic differentiation of mesenchymal stem cells was recorded in the 3-dimensional scaffolds

Other types of nanofibers used in bone regeneration include the natural polymers. Natural polymeric nanofibers, such as poly(caprolactone) (PCL), poly(lactic-co-glycolic acid) (PLGA) poly(L-lactic acid) (PLLA), collagen, gelatine and fibrinogen, are excellent candidates for bone and cartilage tissue engineering applications. These biomaterials possess properties that are useful for bone regeneration, such as biodegradability, flexibility, shape availability and ease of fabrication. Nanoporous polymer matrices can be fabricated via electrospinning, phase

applications (Zhang & Webster, 2009).

compared to 2-dimensional static conventional tissue cultures.

tissue regeneration. Another technique for nanofibrous fabrication is self-assembly. Molecular self-assembly has been applied to produce supramolecular architectures (Silva et al., 2004). This technique produces nanofiber diameters much smaller than those produced using electrospinning. Molecular self-assembly has been less effective in producing macropores for mass transport and cell accommodation. Phase separation techniques have also been also employed to fabricate nanofibers with diameters ranging from 50 – 500 nm and much higher surface –to-volume ratios than produced by other techniques (Chen et al., 2006).

#### **3. Applications of nanotechnology in specific tissue regeneration**

Recent studies have been conducted on the promises and applications of nanotechnology in the regeneration of specific tissues, such as bone, cartilage, vascular and neural tissues.

#### **3.1 Bone and cartilage regeneration**

Various types of traumatic bone and cartilage damage – bone fractures, osteoarthritis, osteoporosis or bone tumours – represent common and significant clinical problems. However, the treatment of such problems with traditional implant materials only lasts 10 – 15 years on average and implant failures originating from implant loosening, inflammation, infection, osteolysis and wear debris frequently occur. There is a very urgent need to develop a new generation of cytocompatible bone and cartilage substitutes to regenerate bone and cartilage tissues at diseased sites that could last the life time of the patient (Zhang & Webster, 2009).

Bone is effectively a nanocomposite that consists of a protein-based soft hydrogel template formed of collagen, non-collagenous proteins such as laminin, fibronectin and vitronectin, water, and hard inorganic components such as hydroxyapatite, calcium and phosphate. Specifically, 70% of the bone matrix is composed of nanocrystalline hydroxyapatite which is typically 20-80 nm long and 2-5 nm thick. Nanostructured bone extracellular matrix closely surrounds and affects adhesion, proliferation and differentiation of mesenchymal stem cells, osteoblasts, osteoclasts and fibroblasts. Moreover, cartilage is a poorly regenerating tissue composed of a small percentage of chondrocytes but dense nanostructured extracellular matrix rich in collagen fibers, proteoglycans and elastin fibers. The limited regenerative properties of cartilage originate from a lack of chondrocyte mobility in the dense extracellular matrix as well as an absence of progenitor cells and the vascular network necessary for efficient tissue repair (Vasita & Katti, 2006). Development of nanotechnology might provide clinical medicine with new prospects in bone and cartilage reconstruction. Nanotechnology employs engineered materials with the smallest functional organization called nanomaterials that are able to interact with biological systems at a nanoscale (El-Sadik et al., 2010). Nanomaterials could be grown or self-assembled to stimulate the dimensions of natural entities, such as collagen fibers. After decreasing material size into nanoscale, dramatically increased surface area, surface roughness and surface area to volume ratios could be created, leading to superior physiochemical properties such as mechanical, electrical, optical, catalytic, magnetic properties. These biomimetic features with the nanostructured extracellular matrix of bone and cartilage played a key role in stimulating cell growth as well as guided tissue regeneration (Jang et al., 2009). Numerous researchers

tissue regeneration. Another technique for nanofibrous fabrication is self-assembly. Molecular self-assembly has been applied to produce supramolecular architectures (Silva et al., 2004). This technique produces nanofiber diameters much smaller than those produced using electrospinning. Molecular self-assembly has been less effective in producing macropores for mass transport and cell accommodation. Phase separation techniques have also been also employed to fabricate nanofibers with diameters ranging from 50 – 500 nm and much higher surface –to-volume ratios than produced by other techniques (Chen et al.,

Recent studies have been conducted on the promises and applications of nanotechnology in the regeneration of specific tissues, such as bone, cartilage, vascular and neural tissues.

Various types of traumatic bone and cartilage damage – bone fractures, osteoarthritis, osteoporosis or bone tumours – represent common and significant clinical problems. However, the treatment of such problems with traditional implant materials only lasts 10 – 15 years on average and implant failures originating from implant loosening, inflammation, infection, osteolysis and wear debris frequently occur. There is a very urgent need to develop a new generation of cytocompatible bone and cartilage substitutes to regenerate bone and cartilage tissues at diseased sites that could last the life time of the patient (Zhang

Bone is effectively a nanocomposite that consists of a protein-based soft hydrogel template formed of collagen, non-collagenous proteins such as laminin, fibronectin and vitronectin, water, and hard inorganic components such as hydroxyapatite, calcium and phosphate. Specifically, 70% of the bone matrix is composed of nanocrystalline hydroxyapatite which is typically 20-80 nm long and 2-5 nm thick. Nanostructured bone extracellular matrix closely surrounds and affects adhesion, proliferation and differentiation of mesenchymal stem cells, osteoblasts, osteoclasts and fibroblasts. Moreover, cartilage is a poorly regenerating tissue composed of a small percentage of chondrocytes but dense nanostructured extracellular matrix rich in collagen fibers, proteoglycans and elastin fibers. The limited regenerative properties of cartilage originate from a lack of chondrocyte mobility in the dense extracellular matrix as well as an absence of progenitor cells and the vascular network necessary for efficient tissue repair (Vasita & Katti, 2006). Development of nanotechnology might provide clinical medicine with new prospects in bone and cartilage reconstruction. Nanotechnology employs engineered materials with the smallest functional organization called nanomaterials that are able to interact with biological systems at a nanoscale (El-Sadik et al., 2010). Nanomaterials could be grown or self-assembled to stimulate the dimensions of natural entities, such as collagen fibers. After decreasing material size into nanoscale, dramatically increased surface area, surface roughness and surface area to volume ratios could be created, leading to superior physiochemical properties such as mechanical, electrical, optical, catalytic, magnetic properties. These biomimetic features with the nanostructured extracellular matrix of bone and cartilage played a key role in stimulating cell growth as well as guided tissue regeneration (Jang et al., 2009). Numerous researchers

**3. Applications of nanotechnology in specific tissue regeneration** 

2006).

& Webster, 2009).

**3.1 Bone and cartilage regeneration** 

fabricated cytocompatible biomimetic nanomaterial scaffolds encapsulating cells, such as stem cells, chondrocytes and osteoblasts. In addition, to the dimensional similarity to bone/cartilage tissue, nanomaterials also exhibited unique surface properties, such as surface topography, surface chemistry, surface wettabilty and surface energy, due to their significantly increased surface area and roughness compared to conventional or micron structured materials. As is known, material surface properties mediate specific protein adsorption and bioactivity, such as fibronectin, vitronectin and laminin, before cells adhere on implants, further, they regulate cell behaviour and dictate tissue regeneration. Furthermore, an important criterion for designing orthopaedic implant materials is the formation of sufficient osseointegration between synthetic materials and bone tissue. Studies have demonstrated that nanostructured materials with cell-favourable surface properties could promote greater amounts of specific protein interactions to more efficiently stimulate new bone growth compared to conventional materials (Webster et al., 2001). This is one of the underlying reasons that nanomaterials are superior to conventional materials for bone growth. Therefore, by controlling surface properties, various nanophase ceramic, polymer, metal and composite scaffolds have been designed for bone/cartilage tissue engineering applications (Zhang & Webster, 2009).

There have been significant advances in the development of bone scaffolds with various compositions and 3 dimensional configurations using a variety of techniques such as the electrospining process for the fabrication of nanofibrous matrices. Several studies have reported the performance of nanofibrous materials in guiding cells to initially adhere to, and spread over, the nanostructures, as well as triggering them to secrete appropriate extracellular matrix molecules targeted to the bone and cartilage tissues. The boneassociated cells and the progenitor/stem cells showed initial responses which were anchorage-dependant. The nanofibrous substratum provided favourable conditions for cell anchorage and growth. Further osteoblastic differentiation and mineralization have also been reported to be regulated in a positive manner on nanofibrous surfaces (Woo et al., 2007). One particular requirement of bone tissue regeneration was that the scaffold should be porous, to incorporate large number of cells. The 3-dimensional scaffolds provided the necessary support for bone cells to attach, grow and differentiate and defined the overall shape of a bone tissue cultured transplant (Jang et al., 2009). Nanofibrous and nanotubular scaffolds were fabricated to mimic collagen fibers in bone and cartilage. Natural collagen is a triple helix self assembled into nanofibers of 300 nm in length and 1.5 nm in diameter. A new nanofiber composite was designed with the same self-assembly pattern as collagen and hydroxyapatite crystals in bone by directly nucleating and aligning the hydroxyapatite on the long axis of a nanofiber. Mesenchymal stem cell behaviour on self-assembled peptide amphiphile nanofiber scaffolds was investigated. Significantly enhanced osteogenic differentiation of mesenchymal stem cells was recorded in the 3-dimensional scaffolds compared to 2-dimensional static conventional tissue cultures.

Other types of nanofibers used in bone regeneration include the natural polymers. Natural polymeric nanofibers, such as poly(caprolactone) (PCL), poly(lactic-co-glycolic acid) (PLGA) poly(L-lactic acid) (PLLA), collagen, gelatine and fibrinogen, are excellent candidates for bone and cartilage tissue engineering applications. These biomaterials possess properties that are useful for bone regeneration, such as biodegradability, flexibility, shape availability and ease of fabrication. Nanoporous polymer matrices can be fabricated via electrospinning, phase

Novel Promises of Nanotechnology for Tissue Regeneration 463

artery. They provided an excellent architecture for endothelial and smooth muscle cell adhesion and proliferation. The aligned fibers affected the behaviour of the smooth muscle cells, and the cytoskeleton is organized to follow the direction of the nanofibers (Xu et al., 2004). Electrospun nanofibers fabricated from natural polymers have been established to develop constructs for vascular tissue regeneration. Electrospun collagen and elastin nanofibers were shown to be good scaffolding systems for the engineering of artificial blood vessels (Boland et al., 2004). Another polymer that promoted the endothelial and vascular smooth muscle cell proliferation was the biodegradable poly(lactic-co-glycolic acid) (PLGA), which produced vascular grafts with nanometer surface features. These nanostructures enhanced fibronectin and vitronectin adsorption from serum leading to better vascular cell responses (Miller et al., 2007). Moreover, self-assembled peptides have been fabricated into scaffolds that mimic the vascular basement membrane with excellent cytocompatability. These peptide scaffolds promote endothelialisation and enhance nitric oxide release and laminin and collagen IV deposition by the endothelial cell monolayer (Genove et al., 2005). Titanium nanostructures have been reported to enhance vascular cell adhesion and proliferation greatly. Competitive endothelial cell functions were promoted over that of vascular smooth muscle cells, solving the problem of the overgrowth of smooth muscle cells

Nanostructure designs have been shown to promote the functional performance of neuronal cells and neural tissue repair. They possess the necessary cytocompatibilty properties for improved neuronal growth, mechanical properties that last long enough to physically support neural tissue regeneration, and electrical properties that stimulate and control neuron behaviour and guide neural tissue repair. Biodegradable and biocompatible novel nanofibers and nanotubes have been fabricated with controlled architecture and components and efficient topography; they promoted neural tissue regeneration. Nanofibrous poly (L-lactic acid) (PLLA) and poly (caprolactone) (PCL) scaffolds designed via electrospining and phase separation demonstrated significant cytocompatibility properties useful for neural tissue regeneration. Incorporation of laminin into the nanofibers created a biomimetic scaffolds for peripheral nerve repair as laminin is an extracellular protein that promotes neurite outgrowth (Koh et al., 2008). Another example for the addition of laminin onto the poly (L-lactic acid) (PLLA) nanofibers was investigated for the culture of the tissues of rat dorsal root ganglia (Patel et al., 2007). Cultures revealed significant longer neurite length more than those cultured on poly (L-lactic acid) (PLLA) nanofibers without laminin. These findings demonstrated the advantages of biosynthetic nanomaterials over the synthetic ones. Moreover, the topography of the electrospun nanofibers scaffolds affected the behaviour of the cultured dorsal root ganglia. Significant extension and elongation of neurites were shown on aligned fibers compared with cultured on randomly oriented nanofibers. The neurites grew in a radial manner on the aligned nanofibers. Those that grew in the direction of the fibers had a faster growth rate than the others indicating that the aligned nanofibrous scaffolds served in guiding neurite

Electrospun Chitosan on poly(caprolactone) (PCL) nanofibrous scaffolds provided excellent mechanical properties that enhanced Schwann cell proliferation (Zhang & Webster, 2009).

in vascular stents (Choudhary et al., 2007).

orientation and cell alignment (Chow et al., 2007).

**3.3 Neural tissue regeneration** 

separation, particulate leaching, chemical etching and 3 dimensional printing techniques (Zhang & Webster, 2009). Poly(caprolactone) (PCL) was first suggested to be a degradable nanofiber matrix for bone regeneration, and it demonstrated good support of the rat bone marrow stromal cells and *in vitro* matrix formation at 4 weeks, including collagen I and calcium phosphate (Yoshimoto et al., 2003). A cell-nanofiber construct was implanted in rat omenta for 4 weeks (Shin et al., 2004). It revealed the formation of collagen I and mineralization similar to bone like extracellular matrix, highlighting its usefulness in bone tissue regeneration. A combination of degradable polymeric nanofibers with bioactive inorganic metals was proved to enhance osteogenic differentiation and calcification of bone matrix. The inorganic phase improved the biological properties of polymers in the bone forming process. Gelatin-hydroxyapatite nanofibers was fabricated (Kim et al., 2005). Hydroxyapatite nanocrystals were distributed in the gelatin matrix and produced an organized hybrid matrix. This composite enhanced osteoblastic differentiation and could be applied usefully in dentistry. In a similar way, collagen-hydroxyapatite (Song et al., 2008) and chitosan-hydroxyapatite (Zhang et al., 2008) nanofibers were generated mimicking the extracellular matrices.

An additional excellent choice of nanomaterials for the reconstruction of bone tissue was the bone-bioactive inorganics such as bioactive glass, ceramics and calcium phosphates. Silica based sol-gel glass mixed with a polymer binder was generated into a nanofibrous mesh by an electrospining technique. Fibers ranging from 84 nm to 640 nm in size were produced (Kim et al., 2006). The large surface area of the nanofibers, and the consequent ionic reaction with the surrounding medium, induced the formation of a bone mineral-like apatite phase on their surfaces. Osteogenic proliferation and differentiation of rat mesenchymal stem cells were found to be enhanced on the bioactive glass nanofiber substrates more than on conventional bioactive glass. Nanophase metals were investigated for orthopaedic tissue regeneration. They are characterized by the presence of more particle boundaries at their surfaces than the conventional micron metals. Linear patterns of nano-features of titanium were created via electron beam evaporation. These patterns induced greater osteoblast adhesion than the micron-rough regions and guided osteoblast morphology and alignment. Highly porous titanium dioxide nanotube layers were fabricated on titanium by anodization. Titanium was anodized electrochemically in dilute hydrofluoric acid electrolyte solutions to produce nanotubes with diameters of 100 nm and lengths of 500 nm into the titanium dioxide layers of titanium. Nanotubular anodized titanium greatly improved osteoblastic function and significantly increased chondrocytic adhesion, promoting bone and cartilage cellular growth (Zhang & Webster, 2009).

#### **3.2 Vascular tissue regeneration**

Researchers have come a long way to develop vascular grafts of great efficacy to replace damaged blood vessels, using materials that produce minimal interactions with the inflowing blood and adjacent tissues. Nanomaterials have been found to improve vascular endothelial and smooth muscle functions. Aligned biodegradable poly(L-lactid-co-epsiloncaprolactone) PLLA-CL (75:25) nanofibrous scaffolds have been tested for their ability to fabricate tubular scaffolds for vessels. These nanofibers demonstrated the mechanical strength needed to sustain high pressure of the human circulatory system and the necessary properties that mimic the dimensions of natural extracellular matrix of human coronary

separation, particulate leaching, chemical etching and 3 dimensional printing techniques (Zhang & Webster, 2009). Poly(caprolactone) (PCL) was first suggested to be a degradable nanofiber matrix for bone regeneration, and it demonstrated good support of the rat bone marrow stromal cells and *in vitro* matrix formation at 4 weeks, including collagen I and calcium phosphate (Yoshimoto et al., 2003). A cell-nanofiber construct was implanted in rat omenta for 4 weeks (Shin et al., 2004). It revealed the formation of collagen I and mineralization similar to bone like extracellular matrix, highlighting its usefulness in bone tissue regeneration. A combination of degradable polymeric nanofibers with bioactive inorganic metals was proved to enhance osteogenic differentiation and calcification of bone matrix. The inorganic phase improved the biological properties of polymers in the bone forming process. Gelatin-hydroxyapatite nanofibers was fabricated (Kim et al., 2005). Hydroxyapatite nanocrystals were distributed in the gelatin matrix and produced an organized hybrid matrix. This composite enhanced osteoblastic differentiation and could be applied usefully in dentistry. In a similar way, collagen-hydroxyapatite (Song et al., 2008) and chitosan-hydroxyapatite (Zhang et al., 2008) nanofibers were generated mimicking the

An additional excellent choice of nanomaterials for the reconstruction of bone tissue was the bone-bioactive inorganics such as bioactive glass, ceramics and calcium phosphates. Silica based sol-gel glass mixed with a polymer binder was generated into a nanofibrous mesh by an electrospining technique. Fibers ranging from 84 nm to 640 nm in size were produced (Kim et al., 2006). The large surface area of the nanofibers, and the consequent ionic reaction with the surrounding medium, induced the formation of a bone mineral-like apatite phase on their surfaces. Osteogenic proliferation and differentiation of rat mesenchymal stem cells were found to be enhanced on the bioactive glass nanofiber substrates more than on conventional bioactive glass. Nanophase metals were investigated for orthopaedic tissue regeneration. They are characterized by the presence of more particle boundaries at their surfaces than the conventional micron metals. Linear patterns of nano-features of titanium were created via electron beam evaporation. These patterns induced greater osteoblast adhesion than the micron-rough regions and guided osteoblast morphology and alignment. Highly porous titanium dioxide nanotube layers were fabricated on titanium by anodization. Titanium was anodized electrochemically in dilute hydrofluoric acid electrolyte solutions to produce nanotubes with diameters of 100 nm and lengths of 500 nm into the titanium dioxide layers of titanium. Nanotubular anodized titanium greatly improved osteoblastic function and significantly increased chondrocytic adhesion,

promoting bone and cartilage cellular growth (Zhang & Webster, 2009).

Researchers have come a long way to develop vascular grafts of great efficacy to replace damaged blood vessels, using materials that produce minimal interactions with the inflowing blood and adjacent tissues. Nanomaterials have been found to improve vascular endothelial and smooth muscle functions. Aligned biodegradable poly(L-lactid-co-epsiloncaprolactone) PLLA-CL (75:25) nanofibrous scaffolds have been tested for their ability to fabricate tubular scaffolds for vessels. These nanofibers demonstrated the mechanical strength needed to sustain high pressure of the human circulatory system and the necessary properties that mimic the dimensions of natural extracellular matrix of human coronary

extracellular matrices.

**3.2 Vascular tissue regeneration** 

artery. They provided an excellent architecture for endothelial and smooth muscle cell adhesion and proliferation. The aligned fibers affected the behaviour of the smooth muscle cells, and the cytoskeleton is organized to follow the direction of the nanofibers (Xu et al., 2004). Electrospun nanofibers fabricated from natural polymers have been established to develop constructs for vascular tissue regeneration. Electrospun collagen and elastin nanofibers were shown to be good scaffolding systems for the engineering of artificial blood vessels (Boland et al., 2004). Another polymer that promoted the endothelial and vascular smooth muscle cell proliferation was the biodegradable poly(lactic-co-glycolic acid) (PLGA), which produced vascular grafts with nanometer surface features. These nanostructures enhanced fibronectin and vitronectin adsorption from serum leading to better vascular cell responses (Miller et al., 2007). Moreover, self-assembled peptides have been fabricated into scaffolds that mimic the vascular basement membrane with excellent cytocompatability. These peptide scaffolds promote endothelialisation and enhance nitric oxide release and laminin and collagen IV deposition by the endothelial cell monolayer (Genove et al., 2005). Titanium nanostructures have been reported to enhance vascular cell adhesion and proliferation greatly. Competitive endothelial cell functions were promoted over that of vascular smooth muscle cells, solving the problem of the overgrowth of smooth muscle cells in vascular stents (Choudhary et al., 2007).

#### **3.3 Neural tissue regeneration**

Nanostructure designs have been shown to promote the functional performance of neuronal cells and neural tissue repair. They possess the necessary cytocompatibilty properties for improved neuronal growth, mechanical properties that last long enough to physically support neural tissue regeneration, and electrical properties that stimulate and control neuron behaviour and guide neural tissue repair. Biodegradable and biocompatible novel nanofibers and nanotubes have been fabricated with controlled architecture and components and efficient topography; they promoted neural tissue regeneration. Nanofibrous poly (L-lactic acid) (PLLA) and poly (caprolactone) (PCL) scaffolds designed via electrospining and phase separation demonstrated significant cytocompatibility properties useful for neural tissue regeneration. Incorporation of laminin into the nanofibers created a biomimetic scaffolds for peripheral nerve repair as laminin is an extracellular protein that promotes neurite outgrowth (Koh et al., 2008). Another example for the addition of laminin onto the poly (L-lactic acid) (PLLA) nanofibers was investigated for the culture of the tissues of rat dorsal root ganglia (Patel et al., 2007). Cultures revealed significant longer neurite length more than those cultured on poly (L-lactic acid) (PLLA) nanofibers without laminin. These findings demonstrated the advantages of biosynthetic nanomaterials over the synthetic ones. Moreover, the topography of the electrospun nanofibers scaffolds affected the behaviour of the cultured dorsal root ganglia. Significant extension and elongation of neurites were shown on aligned fibers compared with cultured on randomly oriented nanofibers. The neurites grew in a radial manner on the aligned nanofibers. Those that grew in the direction of the fibers had a faster growth rate than the others indicating that the aligned nanofibrous scaffolds served in guiding neurite orientation and cell alignment (Chow et al., 2007).

Electrospun Chitosan on poly(caprolactone) (PCL) nanofibrous scaffolds provided excellent mechanical properties that enhanced Schwann cell proliferation (Zhang & Webster, 2009).

Novel Promises of Nanotechnology for Tissue Regeneration 465

air, making them biologically inert. Another technique uses large protein molecules such as bovine serum albumin to could slow the photo-oxidation of the core. Moreover, labelling quantum dots with biomolecules such as arginine-glycine-aspartic acid removed all the toxic effects on cultured stem cells (Solanki et al., 2008). It is recommended to study the appropriate properties and concentrations of different nanoparticles used in cultured and transplanted cells and their safety limits and to deeply understand the physicochemical, molecular and physiological processes of nanomaterials before

Nanotechnology has shown great potential for numerous tissue regeneration applications. Nanomaterials have achieved one of the major challenges of tissue regeneration which is mimicking the architecture of natural extracellular matrix. Designed nanostructures such as nanoparticles, nanosurfaces and nanoscaffolds have been used to promote stem cell cultures which will speed up understanding, controlling and guiding tissue regeneration studies of different tissues, such as bone, cartilage, vascular and neural tissues. It is suggested that the creation of such nanostructures would advance greatly the field of tissue regeneration. However, nanomaterials require more testing and investigations before full use in human tissue repair. Further understanding of their interactions with biological systems is still

Au, K.; Liao, S.; Lee, Y. et al. (2009). Effects of Iron Oxide Nanoparticles on Cardiac

Bacakova, L.; Grausova, L.; Vacik, J. et al. (2007). Improved Adhesion and Growth of

Biggs, M.J.P.; Richards, R.G.; McFarlane, S.; Wilkinson, C.D.W.; Oreffo, R.O.C. & Dalby M. J.

Boland, E.D.; Matthews, J.A.; Pawlowski, K.J. et al. (2004). Electrospinning Collagen and

Boudreau, N.J. & Jones, P.L. (1999). Extracellular Matrix and Integrin Signalling: The Shape

Bulte, J.W.M. & Kraitchman, D.L. (2004). Monitoring Cell Therapy Using Iron Oxide MR Contrast Agents. *Curr Pharm Biotechnol,* Vol.5, No.6, (2004), pp. 567-584

of Things to Come. *Biochem J,* Vol.339, No.3, (1999), pp. 481-488

*Med Devices,* Vol.6, No.5, (September 2009), pp. 515-532

*Interface,* Vol.5, (2008), pp. 1231-1242

Differentiation of Embryonic Stem Cells. *Biochem Biophys Res Commun,* Vol.379,

Human Osteoblast-Like MG 63 Cells on Biomaterials Modified with Carbon Nanoparticles. *Diamond Relat Mater,* Vol.16, No.12 (December 2007), pp. 2133-2140 Baker, B.M.; Handorf, A.M.; Ionescu, L.C.; Li, W. & Mauck, R.L. (2009). New Directions in

Nanofibrous Scaffolds for Soft Tissue Engineering and Regeneration. *Expert Rev* 

(2008). Adhesion Formation of Primary Human Osteoblasts and The Functional Response of Mesenchymal Stem Cells to 330? nm Deep Microgrooves. *J R Soc* 

Elastin: Preliminary Vascular Tissue Engineering. *Front Biosci,* Vol.9, (2004), pp.

introducing them into the human bodies.

**5. Conclusion** 

needed.

**6. References** 

(2009), pp. 898-903

1422-1432

Chitosan micro and nanofiber mesh tubes have also been investigated for nerve reconstruction (Wang et al., 2008). The authors observed early recovery of sensory functions and elongation of the regenerating axons in 10 mm rat sciatic nerve gap after implantation of the nanofiber mesh tubes. Covalent binding of synthetic and natural materials have been demonstrated in the conjugation of collagen onto a copolymer of methyl methacrylate and acrylic acid electrospun nanofibers (Cao et al., 2009). Increased neurite length of cortical neural stem cells, in proportion to collagen content, was found, indicating that this combination improved the attachment and viability of the cultured neural stem cells. Peptide nanofibrous scaffolds fabricated by self-assembly induced favourable neural cell responses and enhanced neuronal cell functions, outgrowth and functional synapse formation (Zhang & Webster, 2009). Other types of scaffolds are the carbon nanotubes and nanofibers. They were found to guide axon regeneration and improve neural activity as a result of good electrical conductivity, strong mechanical properties and their similar nanoscale dimensions to neurites. Multiwalled carbon nanotubes have been applied for the growth of neurons: a 200 % increase in total neurite length and a 300 % increase in the number of branches and neurites have been demonstrated. In addition, decreased astrocyte proliferation, and consequent decreased glial scar tissue formation, was shown on carbon nanofibers with a polymer composite. Moreover, it was found that astrocytes attached and proliferated less on carbon nanofibers with the smallest nanometer diameter and the highest surface energy (Mckenzie et al., 2004). Carbon nanofibers were shown to limit astrocyte functions, leading to decreased glial scar tissue formation which is essential for increased neuronal implant efficacy.

#### **4. Safety issues involved in the use of nanotechnology**

Despite the wide range of applications of nanotechnology in the tissue regeneration studies, still there is a lack of information concerning the influence of nanomaterials on human health. Data available for the safety of nanomaterials, particularly in the field of tissue regeneration, are limited and the mechanisms of their toxicity are still poorly understood. Several studies indicated that a small size, a large surface area and the ability to generate reactive oxygen species increase the potential of nanomaterials to induce cell injury. However, other studies have indicated that, for example, ceramic nanoparticles were safer to osteoblasts than conventional ceramic microparticles. On the other hand, cellular uptake of nanoparticles and their effects on the physiological processes of the cells and their organelles should be deeply investigated before such materials are applied to human tissues. It has been shown, for example, that degradation of nanomaterials used in artificially engineered joints produced toxic responses due to the use of heavy metals such as iron, nickel and cobalt catalysts (Zang & Webster, 2009).

Recent researches in the field of tracking the engrafted stem cells have demonstrated that the safety of quantum dots depends on their physiochemical properties, dose and exposure. Cytotoxicity of quantum dots has been observed owing to the presence of heavy metals such as cadmium and selenium in their cores. Coating the core of quantum dots was recorded to effectively reduce their toxicity to a significant level. Several strategies have been applied to decrease the toxicity of quantum dots. Coating the core with a shell of zinc sulphide reduces the toxicity by blocking the oxidation of the core by air, making them biologically inert. Another technique uses large protein molecules such as bovine serum albumin to could slow the photo-oxidation of the core. Moreover, labelling quantum dots with biomolecules such as arginine-glycine-aspartic acid removed all the toxic effects on cultured stem cells (Solanki et al., 2008). It is recommended to study the appropriate properties and concentrations of different nanoparticles used in cultured and transplanted cells and their safety limits and to deeply understand the physicochemical, molecular and physiological processes of nanomaterials before introducing them into the human bodies.

#### **5. Conclusion**

464 Tissue Regeneration – From Basic Biology to Clinical Application

Chitosan micro and nanofiber mesh tubes have also been investigated for nerve reconstruction (Wang et al., 2008). The authors observed early recovery of sensory functions and elongation of the regenerating axons in 10 mm rat sciatic nerve gap after implantation of the nanofiber mesh tubes. Covalent binding of synthetic and natural materials have been demonstrated in the conjugation of collagen onto a copolymer of methyl methacrylate and acrylic acid electrospun nanofibers (Cao et al., 2009). Increased neurite length of cortical neural stem cells, in proportion to collagen content, was found, indicating that this combination improved the attachment and viability of the cultured neural stem cells. Peptide nanofibrous scaffolds fabricated by self-assembly induced favourable neural cell responses and enhanced neuronal cell functions, outgrowth and functional synapse formation (Zhang & Webster, 2009). Other types of scaffolds are the carbon nanotubes and nanofibers. They were found to guide axon regeneration and improve neural activity as a result of good electrical conductivity, strong mechanical properties and their similar nanoscale dimensions to neurites. Multiwalled carbon nanotubes have been applied for the growth of neurons: a 200 % increase in total neurite length and a 300 % increase in the number of branches and neurites have been demonstrated. In addition, decreased astrocyte proliferation, and consequent decreased glial scar tissue formation, was shown on carbon nanofibers with a polymer composite. Moreover, it was found that astrocytes attached and proliferated less on carbon nanofibers with the smallest nanometer diameter and the highest surface energy (Mckenzie et al., 2004). Carbon nanofibers were shown to limit astrocyte functions, leading to decreased glial scar tissue formation which is essential for increased

neuronal implant efficacy.

**4. Safety issues involved in the use of nanotechnology** 

as iron, nickel and cobalt catalysts (Zang & Webster, 2009).

Despite the wide range of applications of nanotechnology in the tissue regeneration studies, still there is a lack of information concerning the influence of nanomaterials on human health. Data available for the safety of nanomaterials, particularly in the field of tissue regeneration, are limited and the mechanisms of their toxicity are still poorly understood. Several studies indicated that a small size, a large surface area and the ability to generate reactive oxygen species increase the potential of nanomaterials to induce cell injury. However, other studies have indicated that, for example, ceramic nanoparticles were safer to osteoblasts than conventional ceramic microparticles. On the other hand, cellular uptake of nanoparticles and their effects on the physiological processes of the cells and their organelles should be deeply investigated before such materials are applied to human tissues. It has been shown, for example, that degradation of nanomaterials used in artificially engineered joints produced toxic responses due to the use of heavy metals such

Recent researches in the field of tracking the engrafted stem cells have demonstrated that the safety of quantum dots depends on their physiochemical properties, dose and exposure. Cytotoxicity of quantum dots has been observed owing to the presence of heavy metals such as cadmium and selenium in their cores. Coating the core of quantum dots was recorded to effectively reduce their toxicity to a significant level. Several strategies have been applied to decrease the toxicity of quantum dots. Coating the core with a shell of zinc sulphide reduces the toxicity by blocking the oxidation of the core by Nanotechnology has shown great potential for numerous tissue regeneration applications. Nanomaterials have achieved one of the major challenges of tissue regeneration which is mimicking the architecture of natural extracellular matrix. Designed nanostructures such as nanoparticles, nanosurfaces and nanoscaffolds have been used to promote stem cell cultures which will speed up understanding, controlling and guiding tissue regeneration studies of different tissues, such as bone, cartilage, vascular and neural tissues. It is suggested that the creation of such nanostructures would advance greatly the field of tissue regeneration. However, nanomaterials require more testing and investigations before full use in human tissue repair. Further understanding of their interactions with biological systems is still needed.

#### **6. References**


Novel Promises of Nanotechnology for Tissue Regeneration 467

El-Sadik, A.O.; El-Ansary, A. & Sabry, S.M. (2010). Nanoparticle-Labeled Stem Cells: A

Emerit, J.C.; Beaumount; C. & Trivin, F. (2001). Iron Metabolism, Free Radicals, and Oxidative Injury. *Biomed Pharmacother,* Vol.55, No.6, (2001), pp. 333-339 Gao, J. & Xu, B. (2009). Applications of Nanomaterials Inside Cells. *Nanotoday,* Vol.4, (2009),

Gelain, F.; Bottai, D.; Vescovi, A. & Zhang, S. (2006). Designer Self-Assembling Peptide

Genove, E.;Shen, C.; Zhang, S. & Semino, C.E. (2005). The Effect of Functionalized Self-

Grausova, L.; Kromka, A.; Bacakova, L.; Potocky, S.; Vanecek, M. & Lisa, V. (2008). Bone

(2010), pp. 9-16

(2004), pp. 119-125

1988-1994

1529-1535

*PLoS ONE,* Vol.1, No.1, (2006), e119

*Biomaterials,* Vol.26, No.16, (June 2005), pp. 3341-3351

pp. 37-51

Novel Therapeutic Vehicle. *Clinical Pharmacology: Advances and Applications,* Vol.2,

Nanofiber Scaffolds for Adult Mouse Neural Stem Cell 3-Dimensional Cultures.

Assembling Peptide Scaffolds on Human Aortic Endothelial Cell Function.

and Vascular Endothelial Cells in Cultures on Nanocrystalline Diamond Films. *Diamond and Related Materials,* Vol.17, No.7-10, (July-October 2008), pp. 1405-1409 Hu, Y.; Cai, K.; Luo, Z. et al. (2009). Surface Mediated in Situ Differentiation of

Mesenchymal Stem Cells on Gene-Functionalized Titanium Films Fabricated by Layer-by- Layer Technique. *Biomaterials,* Vol.30, No.21, (July 2009), pp. 3626-3635 Huang, D.; Chung, T.; Hung, Y. et al. (2008). Internalization of Mesoporous Silica

Nanoparticles Induces Transient but not Sufficient Osteogenic Signals in Human Mesenchymal Stem Cells. *Toxicol Appl Pharmacol,* Vol.231, (2008), pp. 208-215 Ito, A.; Hibino, E.; Honda, H. et al. (2004). A New Methodology of Mesenchymal Stem Cell

Expansion Using Magnetic Nanoparticle. *Biomechemical and Engineering J,* Vol.20,

Nanoparticle Labelled, Engineered, Autologus Bone Marrow Mesenchymal Stem Cells Following Intra-Articular Injection. *Joint Bone Spine,* Vol.75, (2008), pp. 432-438

Adipose Tissue-Derived Stem Cells in a TGF-b1 Loaded Fibrin-Poly(Lactide-

Labelled Bone MarroDerived Neural Stem Cells after Autologous Transplantation

Biomimetics for Guided Tissue Regeneration. *Adv Funct Matr,* Vol.15, (2005), pp.

Nanofibers as a Next-Generation Biomaterial. *Adv Funct Mater,* Vol.16, (2006), pp.

Jang, J.; Castano, O. & Kim, H. (2009). Electrospun Materials as Potential Platforms for Bone Tissue Engineering. *Advanced Delivery Reviews,* Vol.61, (2009), pp. 1065-1083 Jing, X.; Yang, L.; Duan, X. et al. (2008). Invivo MR Imaging Tracking of Magnetic Iron Oxide

Jung, Y.; Chung, Y.; Kim, S.H. et al. (2009). In situ Chondrogenic Differentiation of Human

Caprolactone) Nanoparticulate. *Biomaterials,* Vol.30, (2009), pp. 4657-4664 Kea, Y.; Hu, C.; Jianga, X. et al. (2009). In vivo Magnetic Resonance Tracking of Feridex-

Kim, H.W.; Song, J.H. & Kim, H.E. (2005). Nanofiber Generation of Gelatin-Hydroxyapatite

Kim, H.W.; Kim, H.E. & Knowles, J.C. (2006). Production and Potential of Bioactive Glass

in Rhesus Monkey. *J Neurosci Methods,* Vol.179, (2009), pp. 45-50


Cao, H.; Liu, T. & Chew, S.Y. (2009). The Application of Nanofibrous Scaffolds in Neural

Chang, J.; Su, H. & Hsu, S. (2008). The Use of Peptide- Delivery to Protect Human Adipose-

Chang, J.; Hsu, S. & Su, H. (2009). The Regulation of The Gap Junction of Human

Charest, J.L.; Bryants, L.E.; Garcia, A.J. & King, W.P. (2004). Hot Embossing for Micropatterned Cell Substrates. *Biomaterials,* Vol.25, (2004), pp. 4767-4775 Chen, V.J. & Ma, P.X. (2004). Nano-Fibrous Poly(L-Lactic Acid) Scaffolds with

Chen, V.J.; Smith, L.A. & Ma, P.X. (2006). Bone Regeneration on Computer-Designed Nano-

Chen, H.; Titushkin, I.; Stroscio, M. & Cho, M. (2007). Altered Membrane Dynamics of

Chow, W.N.; Simpson, D.G.; Bigbee, J.W. & Colello, R.J. (2007). Evaluating Neuronal and

Clem, W.C.; Chowdhury, S.; Catledge, S.A. et al., (2008). Mesenchymal Stem Cell Interaction

Dalby, M.J.; Riehle, M.O.; Yarwood, S.J.; Wilkinson, C.D. & Curtis, A.S. (2003b). Nucleus

Dalby, M.J.; Biggs, M. J.; Gadegaard, N.; Kalna, G.; Wilkinson, C.D. & Curtis, A.S. (2007).

Delcroix, G.J.; Jacquart, M.; Lemaire, L. et al., (2009). Mesenchymal and Neural Stem Cells

Migration Potential in Rat Brain. *Brain Res,* Vol.1255, (2009), pp. 18-31

Spinal Cord Injuries. *Neuron Glia Biol,* Vol.3, (2007), pp. 119-126

Topography. *Exp Cell Res,* Vol.284, (2003b), pp. 274-282

*Nanomedicine,* Vol.4, No.3, (April 2009), pp. 247-248

Fibrous Scaffolds. *Biomaterials,* Vol.27, (2006), pp. 3973-3979

Dots. *Biomaterials,* Vol.29, (2008), pp. 925-936

*Biomaterials,* Vol.30, (2009), pp. 1937-1946

Vol.13, (2007), pp. 1421-1430

(2006), pp. 67-72

pp. 1055-1064

2073

Tissue Engineering. *Advanced Drug Delivery Reviews,* Vol.61, No.12, (October 2009),

Derived Adult Stem Cells from Damage Caused by The Internalization of Quantum

Mesenchymal Stem Cells Through The Internalization of Quantum Dots.

Interconnected Spherical Macropores. *Biomaterials,* Vol.25, No.11, (2004), pp. 2065-

Quantum Dot-Conjugated Integrins During Osteogenic Differentiation of Human Bone Marrow Derived Progenitor Cells. *Biophys J,* Vol.92, (2007), pp. 1399-1408 Choudhary, S.; Haberstroh, K.M. & Webster, T.J. (2007). Enhanced Functions of Vascular

Cells on Nanostructured Ti for Improved Stent Applications. *Tissue Engineering,* 

Glial Growth on Electrospun Polarized Matrices: Bridging the Gap in Percussive

with Ultra-Smooth Nanosrtuctured Diamond for Wear-Resistant Orthopaedic Implants. *Biomaterials,* Vol.29, No.24-25, (August-September 2008), pp. 3461-3468 Curtis, A.S.G.; Dalby, M.J. & Gadegaard, N. (2006). Cell Signaling Arising from

Nanotopography: Implications for Nanomedical Devices. *Nanomedicine,* Vol.1,

Alignment and Cell Signaling in Fibroblasts: Response to a Micro-Grooved

Nanotopographical Stimulation of Mechanotransduction and Changes in Interphase Centromere Positioning. *J Cell Biochem,* Vol.100, (2007), pp. 326-338 Dalby, M.J. (2009). Nanostructured Surfaces: Cell Engineering and Cell Biology.

Labelled with HEDP-coated SPIO Nanoparticles: In Vitro Characterization and


Novel Promises of Nanotechnology for Tissue Regeneration 469

Shi, X.; Wang, Y.; Varshney, R.R. et al., (2009). In-vitro Osteogenesis of Synovium Stem

Shin, M.; Yoshimoto, H. & Vacanti, J.P. (2004). In Vivo Bone Tissue Engineering Using

Silva, G.A.; Czeisler, C.; Niece, K.L. et al. (2004). Selective Differentiation of Neural

Smith, I.O.; Liu, X.H.; Smith, L.A. & Ma, P.X. (2010). Nano-Structured Polymer Scaffolds for

Song, J.H.; Kim, H.E. & Kim, H.W. (2008). Electrospun Fibrous Web of Collagen-Apatite

Sykova, E. & Jendelova, P. (2007). Migration, Fate and In vivo Imaging of Adult Stem Cells

Vasita, R. & Katti, D.S. (2006). Nanofibers and Their Applications in Tissue Engineering. *International Journal of Nanomedicine,* Vol.1, No.1, (2006), pp. 15-30, ISSN 11769114 Wang, L. & Li, D. Y. (2003). Mechanical, Electrochemical and Tribological Properties of

Wang, W.; Itoh, S.; Matsuda, A. et al. (2008). Influences of Mechanical Properties and

Webster, T.J.; Schadler, L.S.; Siegel, R.W. & Bizios, R. (2001). Mechanisms of Enhanced

Woo, K.M.; Jun, J.H.; Chen, J.H.; et al. (2007). Nano-Fibrous Scaffolding Promotes Osteoblast Differentiation and Biomineralization. *Biomaterials,* Vol.28, (2007), pp. 335-343 Xu, C.Y.; Inai, R.; Kotaki, M. & Ramakrishna, S. (2004). Aligned Biodegradable Nanofibrous

Yoshimoto, H.; Shin, H.; Terai, H. & Vacanti, A. (2003). Biodegradable Nanofiber Scaffolds

Zhang, Y.; Venugopal, J.R.; El-Turki, A.; Ramakrishna, S.; Su, B. & Lim, C.T. (2008).

for Bone Tissue Engineering. *Biomaterials,* Vol.29, (2008), pp. 4314-4322

*Nanobiotechnol,* Vol.1, No.2, (March 2010), pp. 226-236

in the CNS. *Cell Death Differ,* Vol.14, (2007), pp. 1336-1342

*and Coatings Technology,* Vol.167, No.2-3, (April 2003), pp. 188-196

Regeneration. *J Biomed Mater Res A,* Vol.84, No.A, (2008), pp. 557-566 Webster, T.J.; Ergun, C.; Doremus, R.H.; Siegel, R.W. & Bizios, R. (2000). Enhanced

*Engineering,* Vol.7, No.3, (2001), pp. 291-301, ISSN 10763279

4005

1352-1355

Vol.10, (2004), pp. 33-41

(2008), pp. 2925-2932

(September 2000), pp. 1803-1810

No.5, (February 2004), pp. 877-886

Vol.24, (2003), pp. 2077-2082

Cells Induced by Controlled Release of Bisphosphate Additives from Microspherical Mesoporous Silica Composite. *Biomaterials,* Vol.30, (2009), pp. 3996-

Mesenchymal Stem Cells on a Novel Electrospun Nanofibrous Scaffold. *Tissue Eng,* 

Progenitor Cells by High-Epitope Density Nanofibers. *Science,* Vol.303, (2004), pp.

Tissue Engineering and Regenerative Medicine. *Wiley Interdiscip Rev Nanomed* 

Precipitated Nanocomposite for Bone Regeneration. *J Mater Sci Mater Med,* Vol.19,

Nanocrystalline Surface of Brass Produced by Sandblasting and Annealing. *Surface* 

Permeability on Chitosan Nano/Microfiber Mesh Tubes as a Scaffold for Nerve

Functions of Osteoblasts on Nanophase Ceramics. *Biomaterials,* Vol.21, No.17,

Osteoblast Adhesion on Nanophase Alumina Involve Vitronectin. *Tissue* 

Structure: A Potential Scaffold for Blood Vessel Engineering. *Biomaterials,* Vol.25,

by Electrospinning and its Potential for Bone Tissue Engineering. *Biomaterials,* 

Electrospun Biomimetic Nanocomposite Nanofibers of Hydroxyapatite/Chitosan


Koh, H.S.; Yong, T.; Chan, C.K. & Ramakrishna, S. (2008). Enhancement of Neurite

Kommireddy, D.S.; Ichinose, I.; Lvov, Y.M. & Mills, D.K. (2005). Nanoparticle Thin Films:

Kommireddy, D.S.; Sriram, S.M.; Lvov, Y.M. & Mills, D.K. (2006). Stem Cell Attachment to

Kubo, K.; Tsukimura, N.; Iwasa, F. et al. (2009). Cellular Behavior on TiO2 Nanonodular

Lenhert, S.; Meier, M.B.; Meyer, U.; Chi, L. & Wiesmann, H.P. (2005). Osteoblast Alignment,

Lin, S.; Xie, X. & Patel, M.R. (2007). Quantum Dot Imaging for Embryonic Stem Cells. *BMC* 

Liu, H. & Webster, T.J. (2007). Nanomedicine for Implants: A Review of Studies and

Mckenzie, J.L.; Waid, M.C.; Shi, R. & Webster, T.J. (2004). Decreased Functions of Astrocytes

Matsuzaka, K.; Walboomers, X.F.; Yoshinari, M.; Inoue, T. & Jansen, J.A. (2003). The

Miller, D.C.; Haberstroh, K.M. & Webster, T.J. (2007). PLGA Nanometer Surface Features

Murthy, S.K. (2007). Nanoparticles in Modern Medicine: State of the Art and Future Challenges. *International Journal of Nanomedicine,* Vol.2, (2007), pp. 129-141 Ogawa, T.; Saruwatari, L.; Takeuchi, K.; Aita, H. & Ohno, N. (2008). Ti Nano-Nodular

Oh, S.; Brammer, K.S.; Julie Li, Y.S.; et al. (2009). Stem Cell Fate Dictated Solely by Altered

Patel, S.; Kurpinski, K.; Quigley, R. et al., (2007). Bioactive Nanofibers: Synergistic Effects of

Shah, B.S.; Clark, P.A.; Moioli, E.K.; Stroscio, M.A. & Mao, J.J. (2007). Labelling of

Blodgett Lithography. *Biomaterials,* Vol.26, (2004), pp. 563-570

Surfaces. *Biomaterials,* Vol.24, (2003), pp. 2711-2719

*Mater Res A,* Vol.81, No.3, (June 2007), pp. 678-684

Vol.29, No.26, (September 2008), pp. 3574-3582

No.24, (August 2006), pp. 4296-4303

(October 2009), pp. 5319-5329

*Biotechnol,* Vol.7, (2007), pp. 67

369

pp. 1309-1317

751-756

2130-2135, ISSN 1729-8806

(2007), pp. 2122-2128

pp. 3071-3079

(2005), pp. 286-290

Outgrowth Using Nanostructured Scaffolds Coupled with Laminin. *Biomaterials,* 

Surface Modification for Cell Attachment and Growth. *J Biomed Nanotechnol,* Vol.3,

Layer-by-Layer Assembled TiO2 Nanoparticle Thin Films. *Biomaterials,* Vol.27,

Structures in a Micro-to-Nanoscale Hierarchy Model. *Biomaterials,* Vol.30, No.29,

Elongation and Migration on Grooved Polystrene Surfaces Patterned by Langmuir-

Necessary Experimental Tools. *Biomaterials,* Vol.28, No.2, (January 2007), pp. 354-

on Carbon Nanofiber Materials. *Biomaterials,* Vol.25, No.7-8, (March-April 2004),

Attachment and Growth Behavior of Osteoblast-like Cells on Microtextured

Manipulate Fibronectin Interactions for Improved Vascular Cell Adhesion. *J Biomed* 

Structuring for Bone Integration and Regeneration. *J Dent Res,* Vol.87, (2008), pp.

Nanotube Dimension. *Proc Natl Acad Sci USA,* Vol.106, No.7, (February 2009), pp.

Nanotopography and Chemical Signaling on Cell Guidance. *Nano Lett,* Vol.7,

Mesenchymal Stem Cells by Bioconjugated Quantum Dots. *Nano Lett,* Vol.7, (2007),


**Modeling and Assessment of Regeneration** 

Zhang, L. & Webster, T.J. (2009). Nanotechnology and Nanomaterials: Promises for Improved Tissue Regeneration. *Nanotoday,* Vol.4, No.1, (February 2009), pp. 66-80 **Part 4** 
