**4. Decellularization**

*3.2.3. Application*

262 Regenerative Medicine and Tissue Engineering

presence or absence of crosslinkers.

ease of formability and inherent blood compatibility.

suitable for development of ocular bandage lens [58].

Chitin and chitosan are known for their excellent biological properties, among which the biocompatibility with human cells, the ordered regeneration of wounded tissues, the immu‐ noenhancing activity, the induction of immediate hemostasis, the radical scavenging activity, and the antimicrobial activity. Recent studies indicate that chitin and chitosan are most versatile in drug and gene delivery, elaborated diagnostics, devices for selective recognition of tumor cells, and surgical aids ranging from anti-adhesion gels to coated sterile stents.

As a kind of renewable resource, unmodified chitosan has been widely used in many fields such as pharmaceutical, agriculture, food, and biomedical applications. In order to realize the full potential of chitosan and bring a breakthrough in its broader utilization, attempts have been made to modify chitosan to obtain various derivatives. For the tissue repair and regen‐ eration applications, chitosan can be functionalized by chemical reaction, coupling with specific ligands or moieties, combining with biomacromolecules, and crosslinking in the

Particularly, skin substitute made of chitosan or its derivatives have attracted much attention due to the outstanding characteristics of chitosan, such as biocompatibility, hemostatic activity,

The design of artificial kidney systems has made possible repetitive hemodialysis and the sustaining life of chronic kidney failure patients. Chitosan membranes have been proposed as an artificial kidney membrane because of their suitable permeability and high tensile strength. The most important part of artificial kidney is the semipermeable membrane and so far made from commercial regenerated cellulose and cuprophane. Since the primary action of the cellulose membrane is that of a sieve, there is little selectivity in the separation of two closely related molecules. These novel membranes need to be developed for better control of transport,

A series of membranes prepared from chitin and its derivatives improved dialysis properties. One of the most serious problems of using these artificial membranes is surface induced thrombosis, where heparization of blood is needed to prevent clotting, and people who are liable to internal hemorrhage can be dialysed only at great risk. Hence, these are the most challenging problem still to be resolved in the development of membranes which are inher‐ ently blood compatible. From these point of views, chitosan is hemostatic, i.e., causes clots [57].

Chitosan has replaced the synthetic polymers in opthalmological applications. Chitosan possesses all the characteristics required for an ideal contact lens; optical clarity, mechanical stability, sufficient optical correction, gas permeability, partially towards oxygen, wettability, and immunologically compatibility. Contact lenses are made from partially depolymerized and purified squid pen chitosan by spin casting technology, and these contact lenses are clear, tough, and possess other required physical properties such as modulus, tensile strength, tear strength, elongation, water content, and oxygen permeability. Antimicrobial and wound healing properties of chitosan along with excellent film forming capability make chitosan

antibacterial property, and ability to accelerate the wound-healing process [56].

Autologous grafts are "gold standard" for implantation. However, the most disadvant‐ age of autologous is quantity. The number of autograft does not meet needs of patients. Homograft is greater than autograft but they cannot satisfy needs of patient. Many patients must wait for a long time to take a homogenous organ. Xenograft is the greatest but they can evoke serious immune reaction. So, one method developed to process homograft and xenograft is decellularization. Every tissue/organ concludes cells and extracellular matrix. Cells are structure and functional units of tissue/organ but cells are major antigen of tissue/ organ. Extracellular matrix is many protein, polysaccharide, protoglycan released by cell. ECM plays an important role in mechanical support, signal transportation, adherence of tissue/organ. Decellularization is a multi-step process to remove all cell components from tissue/organ and leave intact ECM. Many decellularization agents were researched such as physical methods, chemical methods and enzyme methods. Every decellularization agent has specific affections of cell and extracellular matrix. So, these agents are combined to make an effective decellularization process which removes all cell components and reverses maximum ECM. Decellularization effectiveness depends on type of tissue/organ. One agent can be a good detergent for decellularizing one tissue but not for another [61]. More‐ over, cell derived ECM can be used as a matrix for cell culture.


**Table 1.** Overview of decellularization methods (Thomas W. Gilbert)

Today, many decellularization grafts were applied in clinical treatment such as acellular valve, vascular and some are commercial include: SurgisSIS (porcine small intestinal submicosa), Alloderm (human dermis), ACell (porcine urinary bladder)… Small intestine contains four layers such as mucosa, submucosa, muscularis externa and serosa. Small intestine submucosa (SIS) is a submucosa tissue between mucosa and muscularis externa. SIS is isolated from small intestine by mechanically removing of internal mucosal layer and outer muscular layer. Then, SIS is processed step-by-step with 0.1% peracid acid, 0.05% gentamycin and sterilized using 2500 kRad gamma irradiation. SIS is consisted of collagen, proteoglycan, glycosaminoglycan, glycoprotein and growth factor (VEGF, FGF-2 …). Most of these components can be preserved in extracellular matrix after decellularizing. In tissue engineering, SIS is used as soft tissue grafts such as vascular, skin or used for reconstructing genitourinary, ligament tissue [62].

#### **4.1. Vascular tissue engineering**

**Method Mode of action Effects on ECM**

Pressure can burst cells and tissue removal

Can cause cell lysis, but more commonly used to facilitate chemical exposure and

Disrupts lipid–lipid and lipid–protein interactions, while leaving protein – protein interactions intact

Solubilize cytoplasmic and nuclear cellular membranes; tend to denature proteins

Chelating agents that bind divalent metallic ions, thereby disrupting cell

Catalyze the hydrolysis of the interior bonds of ribonucleotide and deoxyribonucleotide chains

Catalyze the hydrolysis of the terminal bonds of ribonucleotide and deoxyribonucleotide chains

Sodium deoxycholate More disruptive to tissue structure than SDS

cellular material removal

Alkaline; acid Solubilize cytoplasmic components of cells; disrupts nucleic acids

CHAPS Exhibit properties of non-ionic and ionic detergents

Tri(n-butyl)phosphate Organic solvent that disrupts protein– protein interactions

adhesion to ECM

Trypsin Cleaves peptide bonds on the C-side of Arg and Lys

**Table 1.** Overview of decellularization methods (Thomas W. Gilbert)

hypertonic solutions Cell lysis by osmotic shock

membrane ECM can be disrupted or fracture during rapid freezing

cellular material is removed

Removes GAGs

collagen

detergents

Triton X-100

cellular remnants

immune response

X-200

Aggressive agitation or sonication can disrupt ECM as the

Mixed results; efficiency dependent on tissue, removes GAGs

Removes nuclear remnants and cytoplasmic proteins; tends to disrupt native tissue structure, remove GAGs and damage

Yielded efficient cell removal when used with zwitterionic

Efficient cell removal with ECM disruption similar to that of

Yielded cell removal and mild ECM disruption with Triton

Variable cell removal; loss of collagen content, although effect on mechanical properties was minimal

Efficient for cell lysis, but does not effectively remove the

Prolonged exposure can disrupt ECM structure, removes

Difficult to remove from the tissue and could invoke an

No isolated exposure, typically used with enzymatic methods (e.g., trypsin)

laminin, fibronectin, elastin, and GAGs

eliminates cells Mechanical force can cause damage to ECM

Snap freezing Intracellular ice crystals disrupt cell

264 Regenerative Medicine and Tissue Engineering

Physical

Chemical

Triton X-100

(SDS)

Triton X-200

Ionic detergents

Sodium dodecyl sulfate

Zwitterionic detergents

Sulfobetaine-10 and -16 (SB-10, SB-16)

Hypotonic and

EDTA, EGTA

Enzymatic

Endonucleases

Exonucleases

Mechanical force

Mechanical agitation

Non-ionic detergents

Atherosclerosis is the most dangerous cardiovascular disease. Atherosclerosis is a condition in which vascular wall is harden, thicken because of fatty lipid accumulation. Atherosclerosis is responsible for millions of death all the world every year. In 2004, seventeen million people passed away because of cardiovascular disease all over the world (29% world total death). More than 7 million people are killed by coronary disease (9.6% world total death) (WHO). Heart American Association, in 2006, coronary disease was responsible for 17.6% death in America. One out of six American died for coronary disease [63].

Today, there are some methods to cure atherosclerosis including drug treatment, coronary artery bypass surgery and angioplasty. However, these methods can not cure this condition completely. More than 30% patients don't have qualified autogenous vascular grafts. There‐ fore, artificial grafts are made to overcome some current drawbacks.

Artifical graft can be classified into synthetic and biologic scaffold. Synthetic scaffolds include undegradable polymers such as Dacron, ePTFE and degradable polymers such as polylactic acid (PLA) and polyglycolic acid (PGA). Some advantages of synthetic polymers are easy processing, high initial strength, however, they have some disadvantages such as slow recellularization, poor ability to support remodeling. Biologic vascular grafts are either obtained by manipulating native proteins of vascular vessels such as collagen, elastin… or by decellularizing vascular vessels. Decellularized native vascular vessels have some advantages such as slow cost, reduction of graft rejection and immune reaction [64].

In the early time, decellularization studies were tested without cell-seeding. In 1990, Lantz et al use SIS (small intestine submucosa) as small-diameter arterial graft in18 dogs. 48 weeks after surgery, 28 vascular grafts (75%) were patent and tree dogs can survive from 76 to 82 weeks after implantation. This result suggested that small intestinal submucosa can be used in small diameter vascular tissue engineering [65]. In 2001, Hodde et al demonstrated that porcine SIS ECM extract containing vascular endothelial growth factor (VEGF) with concentration of 0.77 ng VEGF/g SIS [66]. In the same year, Nemcova et al isolated and decellularized SIS from bovine and porcine. Nine decellularized SIS grafts were implanted into femoral arteries of five male mongrel dogs. After 9 weeks, eight grafts remained patent and some kinds of cell such as endothelial cells (ECs), smooth muscle cells (SMCs) invaded into the grafts. No evidence of inflammatory and aneurysmal symptom was observed during the experimental time [67].

However, the main disadvantage of vascular graft is thrombus which ussually occurs imme‐ diately in vascular graft lumen after implantation. This process can lead to graft failure and threaten patients' survival. In order to solve this problem, ECs are used as anti-thrombus agent in vascular graft lumen, additionally, SMCs are used to improve mechanical strength of vascular graft. Broschel et al decellularized and recellularized rat iliac arterial grafts. Iliac arterial grafts were decellualrized by glycerin, SDS for 12 hours. Then, decellularized iliac grafts were recellularized with adult rat heart ECs and implanted to femoral arteries of allogeneic rats without systemic anticoagulation injection. After 4 weeks, 2 of 7 control grafts (29%) were patent (decellularized grafts without recellularization) and 8 of 9 (89%) experi‐ mental grafts (decellularized grafts with recellularization) maintained blood vessel patent [68]. Consequently, this experiment result proved excellent function of ECs in vascular tissue engineering. Some researchers on vascular tissue engineering seeded autologous vascular cells including ECs, SMCs and fibroblasts to make autologous tissue engineered vascular. At the same time, the appearance of bioreactor systems sped up development of vascular tissue engineering. Niklason developed a pulsatile perfusion bioreactor system in 2001. Bioreactor contained a stirbar, a lid for gas exchange and one (or two) silicone tubing(s). Porous vascular scaffolds were threaded over silicone tubing. Bovine aortic SMCs at the passage 2 or 3 were pipetted onto the outer surface of the scaffolds, then bioreactors were slowly rotated and removed to incubator with 10% CO2, 100% humidity, and 37o C. Each silicone tubing was linked to a pulsatile perfusion system operated at 165 beats/minute and 260/-30 mmHg. After 8 weeks culture, under nonpulsatile condition, SMC growth was on the outer surface of tubing scaffold and poorly organized. Under pulsatile condition, SMC distribute homogenously in scaffold wall from outer to inner similar to native structure [69].

Nowaday, some decellularized vascular grafts can be obtained from human. Human umbilical veins and arteries can be used in decellularization experiment. Human umbilical arteries were isolated and completely decellularized by Gui et al in 2009. Decellularized umbilical arteries preserved intact collagen matrix and mechanical properties, burst pressure had no signicant change from native form. Decellularized artery graft remained patent after 8 weeks surgery [70]. In 2005, Joel Daniel et al processed human umbilical vein by automated dissection. Human umbilical cord was inserted a mandrel into vein and frozen to -20 and -80o C, human umbilical cord was maintained for 12 hours at least at this temperature. The mandrel was lathed with rotation speed of 2900 rpm, cutting depth of 750 μm. Then, human umbilical vein was decellularized with 1% (w/v) SDS. The result showed that the treated human umbilical vein contained no cell; burst pressure results were 972.8 ± 133.8 mmHg (972/1082), compliance results were 5.7 ± 1.3% over 80 – 120 mmHg. Vascular smooth muscle cells can adhere, proliferate and migrate on the surface of dHUV [71]. Tran Le Bao Ha et al carried out a research on decellularization method for HUVs. HUVs were isolated by manual dissection. HUVs were decellularized by distilled water or NaCl 3M or SDS 1%. The result confirmed that the combination between SDS 1% (24h) and NaCl 3M (24h) showed the best effective on cellular elimination.

**Figure 6.** Human umbilical cord (a) and human umbilical vien (b)

as endothelial cells (ECs), smooth muscle cells (SMCs) invaded into the grafts. No evidence of inflammatory and aneurysmal symptom was observed during the experimental time [67].

However, the main disadvantage of vascular graft is thrombus which ussually occurs imme‐ diately in vascular graft lumen after implantation. This process can lead to graft failure and threaten patients' survival. In order to solve this problem, ECs are used as anti-thrombus agent in vascular graft lumen, additionally, SMCs are used to improve mechanical strength of vascular graft. Broschel et al decellularized and recellularized rat iliac arterial grafts. Iliac arterial grafts were decellualrized by glycerin, SDS for 12 hours. Then, decellularized iliac grafts were recellularized with adult rat heart ECs and implanted to femoral arteries of allogeneic rats without systemic anticoagulation injection. After 4 weeks, 2 of 7 control grafts (29%) were patent (decellularized grafts without recellularization) and 8 of 9 (89%) experi‐ mental grafts (decellularized grafts with recellularization) maintained blood vessel patent [68]. Consequently, this experiment result proved excellent function of ECs in vascular tissue engineering. Some researchers on vascular tissue engineering seeded autologous vascular cells including ECs, SMCs and fibroblasts to make autologous tissue engineered vascular. At the same time, the appearance of bioreactor systems sped up development of vascular tissue engineering. Niklason developed a pulsatile perfusion bioreactor system in 2001. Bioreactor contained a stirbar, a lid for gas exchange and one (or two) silicone tubing(s). Porous vascular scaffolds were threaded over silicone tubing. Bovine aortic SMCs at the passage 2 or 3 were pipetted onto the outer surface of the scaffolds, then bioreactors were slowly rotated and

to a pulsatile perfusion system operated at 165 beats/minute and 260/-30 mmHg. After 8 weeks culture, under nonpulsatile condition, SMC growth was on the outer surface of tubing scaffold and poorly organized. Under pulsatile condition, SMC distribute homogenously in scaffold

Nowaday, some decellularized vascular grafts can be obtained from human. Human umbilical veins and arteries can be used in decellularization experiment. Human umbilical arteries were isolated and completely decellularized by Gui et al in 2009. Decellularized umbilical arteries preserved intact collagen matrix and mechanical properties, burst pressure had no signicant change from native form. Decellularized artery graft remained patent after 8 weeks surgery [70]. In 2005, Joel Daniel et al processed human umbilical vein by automated dissection. Human

cord was maintained for 12 hours at least at this temperature. The mandrel was lathed with rotation speed of 2900 rpm, cutting depth of 750 μm. Then, human umbilical vein was decellularized with 1% (w/v) SDS. The result showed that the treated human umbilical vein contained no cell; burst pressure results were 972.8 ± 133.8 mmHg (972/1082), compliance results were 5.7 ± 1.3% over 80 – 120 mmHg. Vascular smooth muscle cells can adhere, proliferate and migrate on the surface of dHUV [71]. Tran Le Bao Ha et al carried out a research on decellularization method for HUVs. HUVs were isolated by manual dissection. HUVs were decellularized by distilled water or NaCl 3M or SDS 1%. The result confirmed that the combination between SDS 1% (24h) and NaCl 3M (24h) showed the best effective on cellular

C. Each silicone tubing was linked

C, human umbilical

removed to incubator with 10% CO2, 100% humidity, and 37o

266 Regenerative Medicine and Tissue Engineering

wall from outer to inner similar to native structure [69].

elimination.

umbilical cord was inserted a mandrel into vein and frozen to -20 and -80o

**Figure 7.** HE staining of HUV with different decellularization methods. Control (a), distilled water (b), NaCl 3M (c), SDS 1% 24h (d), SDS 1% 36h (e), SDS 1% 24. and NaCl 3M 24h (f)

#### **4.2. ECM from cultured cells**

One of the most important properties of ECM is its functional diversity. ECM has been reported to support and enhance for adhesion, migration, proliferation of cells as well as to create stem cell niches *in vitro*.. ECM can be harvested from different sources, one of which is from cells under culture condition. When cultured, cells will produce three-dimensional matrix sur‐ rounding themselves. A method is described for generating tissue culture surfaces coated with a human fibroblast-derived ECM [72, 73, 74, 75].

For this purpose, human foreskin fibroblasts are isolated, plated and cultured until third passage (see Figure 8). Fibroblasts are maintained in culture medium until reaching 80% confluency and stimulated to synthesize ECM by culture medium supplemented ascorbic acid. Matrices are denuded of cells and cellular remnants are removed by using Triton X-100, NH4OH and DNase.

Then, ECM coated culture surfaces are tested by staining with PI to access DNA remnant (see Figure 9), with H&E and PAS in order to characterize component of ECM. The results suggest that fibroblast-derived three-dimensional matrix was determined to be free of cellular constituents and still remain attached to the culture surface. The conducted matrices were washed and covered with PBS; and stored at 40 C. Under these conditions, biological activity (for example, induction of cell attachment, proliferation…) was reported to be well-preserved for up to 6 months.

In order to evaluate the quality of the ECM, the test of cell rapid attachment ability is per‐ formed. Cell proliferation on ECM is also assessed. Results showed that the cultured cells attached and proliferated on ECM coated surfaces faster than on ECM non-coated surfaces.

**Figure 8.** Fibroblasts are in cultured surfaces (a) and are stimulated to synthesize ECM (b)

**Figure 9.** ECM stained PI before (a) and after (b) using DNase

**Figure 10.** Culture surface before (a) and after (b) is denuded cells
