Open access peer-reviewed chapter - ONLINE FIRST

Supercritical Carbon Dioxide Facilitated Collagen Scaffold Production for Tissue Engineering

Written By

Periasamy Srinivasan and Dar-Jen Hsieh

Submitted: December 6th, 2021Reviewed: January 3rd, 2022Published: February 7th, 2022

DOI: 10.5772/intechopen.102438

IntechOpen
Collagen BiomaterialEdited by Nirmal Mazumder

From the Edited Volume

Collagen Biomaterial [Working Title]

Dr. Nirmal Mazumder and Dr. Sanjiban Chakrabarty

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Abstract

The rise of tissue engineering and regenerative medicine (TERM) is a developing field that focuses on the advancement of alternative therapies for tissue and organ restoration. Collagen scaffold biomaterials play a vital role as a scaffold to promote cell growth and differentiation to promote the repair and regenerate the tissue lesion. The goal of this chapter will be to evaluate the role of supercritical carbon dioxide extraction technology in the production of collagen scaffold biomaterials from various tissues and organs and relate it to the traditional decellularization techniques in the production of collagen biomaterials for TERM. Therefore, we will study the collagen scaffold biomaterials produced using supercritical carbon dioxide extraction technology and their characteristics, such as chemical-physical properties, toxicity, biocompatibility, in vitro and in vivo bioactivity that could affect the interaction with cells and living system, relative to traditional decellularization technique-mediated collagen scaffolds. Furthermore, the chapter will focus on supercritical carbon dioxide extraction technology for the production of collagen scaffolds biomaterial appropriate for TERM.

Keywords

  • supercritical carbon dioxide extraction technology
  • tissue engineering
  • regenerative medicine
  • biomaterial
  • collagen scaffold
  • biocompatibility

1. Introduction

Tissue engineering advanced from the field of biomaterials development and denotes the practice of combining cells, tissue scaffolds, and bioactive signal molecules. These tissue scaffolds are produced by various decellularization processes, such as chemical and physical methods. Tissue scaffolds, cells, and biologically active signal molecules are the three key elements for tissue and organ reparation. Tissue engineering is defined as “an interdisciplinary field of research that applies the principles of engineering and the life science toward the development of biological substitutes that restore, maintain or improve tissue function” [1]. Regenerative medicine is a wide field that comprises tissue engineering but also integrates research on self-healing in which the body uses its systems, sometimes with help of foreign biological material to reconstruct and rebuild tissues and organs. The terms tissue engineering and “regenerative medicine” have become largely interchangeable, as the field hopes to focus on cures as an alternative for the treatment of complex, mainly chronic diseases (e.g. Diabetic wound healing, burn wounds).

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2. Tissue engineering and regenerative medicine

Tissue engineering and regenerative medicine (TERM) have been projected and established for almost 30 years. Though many fruitful challenges in tissue regeneration have been attained, TERM is still in its infancy stage and most of the vital questions remain to be answered, including the selection of cell sources, development of tissue-specific materials, and construction of complex organs. The most important is the in vivomechanism of the formation of new tissue and organ employing the tissue-engineered biomaterials, and the process to resemble and transform to native tissue and organ. The subsequent transformation and final destination of the biomaterials remain to be the serious apprehensions in this dynamically emerging field. Addressing these queries is significant to the effectiveness, stability, and security of the clinical application of tissue-engineered biomaterials [2].

Tissue and organ repair remain a clinical issue and challenge. Entirely restoring or regenerating damaged tissues and organs and reestablishing their functions have been a vision of medical society. The emergence of tissue engineering and regenerative medicine (TERM) makes it possible. TERM is a developing field that focuses on the advancement of alternative therapies for tissue and organ restoration [2, 3]. TERM is an extremely multidisciplinary arena, in which bioengineering and medicine unite. It is constructed on integrative approaches using scaffolds, cells, growth factors, nanomedicine, and other techniques to pass on the restrictions that presently exist in the hospitals. Certainly, TERM overall aim is to encourage the formation of new functional tissues, rather than just implanting tissue and bone parts [2]. TERM is a multifaceted science and associates basic sciences such as materials science, biomechanics, cell biology, and medical sciences to comprehend functional tissue and organ restoration and reconstruction. The world’s population is aging and the trend is escalating. There is a severe global shortage of tissues and organs for transplantation. TERM has the potential to meet the requirements of the forthcoming needs of patients [2, 4]. TERM aims to create a three-dimensional (3D) cell-biomaterial composite, that possesses a comparable function as living tissue and organ and is employed to restore or regenerate damaged tissue and organ. The basic condition for the 3D composite is to support cell growth, nutrition and waste transport and gas exchange. TERM typically employs the following strategies, cell-biomaterial composite, in which cell-seeded biomaterials are implanted into the body to restore and regenerate tissues and organs; stem cell transplantation; and biomaterial implanted into the body and undertake the process of tissue integration [3]. Scaffolds are vital for tissue engineering approaches for several reasons; as a three-dimensional structure, they offer volume fill, mechanical integrity and a surface that can afford chemical and architectural guidance for regenerating tissues [5]. The three vital elements in TERM are cells, scaffolds and signals (Figure 1). Several decellularization techniques had been used for the production of collagen scaffolds for TERM application, including the supercritical carbon dioxide (SCCO2) extraction technology to be discussed here in this article.

Figure 1.

The triad of TERM.

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3. Collagen scaffolds-biomaterial for TERM

Collagen-based biomaterial application in the field of TERM has been significantly increasing over the past decades. Collagen owns the main advantages as it is biodegradable, biocompatible, easily available and highly versatile. However, collagen is a protein, therefore it is problematic to sterilize without altering its native structure. Collagen-based biomaterials developed for TERM were intended to provide a functional biomaterial for use in TERM from the laboratory bench to the patient bedside [6]. Collagen is present in all connective tissue and makes it one of the most studied biomolecules of the extracellular matrix (ECM). It is the major component of skin and bone and constitutes approximately 25–35% of mammalian total dry weight [7]. Until now, 29 diverse collagen genotypes have been characterized and all depict a typical triple helix structure. Fiber form of collagens are types I, II, III, V and XI. Collagen molecules are made up of three α chains that assemble due to their molecular structure. Each α chain is made up of more than a 1000 amino acids based on the repeated sequence -Gly-X-Y-. The vital part is the presence of glycine at every third amino acid position to permit for a tight triple-helical packaging of the three α polypeptide chains. In the tropocollagen molecule the X and Y positions are mostly filled by proline and 4-hydroxyproline [6, 7]. Though numerous types of collagens (Table 1) have been defined, only a few types are used to yield collagen-based biomaterials. Currently, type I collagen is the gold standard in the field of TERM.

TypeMolecular formulaFormDistribution
I[α1(I)]2α2(I)FibrilBone, skin, tendons, ligaments, cornea
II[α1(II)]3FibrilCartilage, intervertebral disc, notochord, vitreous humor in the eye
III[α1(III)]3FibrilSkin, blood vessels
V[α1(V)]2α2(V) and α1(V)α2(V)α3(V)Fibril (assemble with type I)idemas type I
XIα1(XI)α2(XI)α3(XI)Fibril (assemble with type II)idemas type II
IXα1(IX)α2(IX)α3(IX)Lateral association with type II fibrilCartilage
XII[α1(XII)]3Lateral association with type I fibrilTendons, ligaments
IV[α1(IV)]2α2(IV)Sheet-like networkBasal lamina
VII[α1(VII)]3Anchoring fibrilsBeneath stratified squamous epithelia

Table 1.

Collagen types, forms and distribution [6].

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4. Collagen immunogenicity and biocompatibility

Medical application of collagen biomaterial needs to make a clear difference between immunogenicity and antigenicity. Immunogenicity is triggering an immune response; however, antigenicity denotes the interaction between the antibodies and the antigenic epitopes. Collagen mediated immune response primarily targets epitopes in the telopeptide region at each end of the tropocollagen molecule. The polymerized collagen fibrils conformity of the helical part and the amino acid sequence on the surface can influence the immunologic profile of the collagen molecule [7]. Type I collagen is an appropriate biomaterial for implantation meanwhile only an insignificant number of people have humoral immunity against type I collagen. In addition, a simple serologic test can validate an allergic reaction in response to type I collagen-based biomaterial. It is most crucial to discuss that collagen immunogenicity which is relevant to collagen molecules that are made up of an acellular ECM and the utmost adverse immune responses that have been come across with an acellular scaffold are not necessarily initiated from the collagen molecule itself. Incomplete decellularization with the presence of remaining oligosaccharide α-Gal and DNA is the common reason for acute immune responses and subsequent acellular ECM rejection [7, 8].

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5. Traditional decellularization of tissues and organs for collagen biomaterial

The traditional decellularization techniques involve long duration and increased cost as well as long-term washing of the tissue material from the residual and traces of the chemicals used. Despite the numerous decellularization process that exists, it is necessary to go through a lot of parameters for multiple reasons in the decellularization process (Table 2). The decellularization process aims to remove the cellular material of the donor, antigens, and potential pathogens. In addition, the most critical issue is to offer the conservation of the structural organization of an ECM with the set of functions inherent in it. Therefore, the optimization of these decellularization methods and the pursuit of improved methods are still ongoing [9]. At present, numerous procedures for decellularization of tissues were employed that include the treatment by detergents such as sodium dodecyl sulfate (SDS), sodium deoxycholate, Triton X-100, etc., and treatment by enzymes such as trypsin, deoxyribonuclease (DNase), and ribonuclease (RNase). Other methods include alkali treatment, as well as cyclic freezing-thawing and high-pressure action up to 1 GPa, which have been tried (Table 3) [9, 10].

Decellularization techniquesAdvantagesDisadvantages
Supercriticalcarbon dioxide extraction technologySupercritical phase pressure disrupts ECM. Uses inert gas (CO2) for cell removal and do not alter ECM’s mechanical propertiesNo known disadvantages. Not yet widely employed for decellularization
Acids and bases(sulfuric acid, ammonium hydroxide, acetic acid, peracetic acid)Disinfects material by entering inside microorganisms and oxidizing enzymesDissociate important molecules in ECM including GAGs, from collagen scaffold
Enzymes(trypsin)Better preservation of GAG contentDisruptive to elastin and collagen and elicits immune response
Non-ionic detergents(Triton X-100)Preserves protein-protein interactions intact and retains sulphated GAG contentDisrupts ECM ultrastructure and owers laminins/fibronectin content
Ionic detergents (sodium dodecyl sulphate (SDS))Effectively removes cells from the tissueDisrupts protein-protein interactions and causes a decrease in GAG content and collagen integrity
Zwitterionic detergents(CHAPS and SB-10/SB-16)Preserves native ECMA high degree of protein denaturation
Solvents(alcohols and acetone)No advantagesCrosslinks and precipitates proteins, including collagen
SonicationEffective cell removalParameters are not well standardized
High-pressure gradient systemEffective cell removalIneffective for densely organized ECM tissues

Table 2.

Decellularization techniques used for tissues, organs and their advantages and disadvantages.

Area of applicationTitleAuthorsSignificance
Bone regenerationDevelopment of a decellularized porcine bone graft by supercritical carbon dioxide extraction technology for bone regeneration.Chen et al. [23]Production and evaluation of biocompatibility of bone graft
Skin regenerationRegenerative porcine dermal collagen matrix developed by supercritical carbon dioxide extraction technology: Role in accelerated wound healing.Wang et al. [33]Wound healing, skin graft and regeneration
Skincare industry and medical applicationsProtocols for accelerated production and purification of collagen scaffold and atelocollagen from animal tissues.Hsieh & Srinivasan [34]Accelerated production and purification of atelocollagen
Experimental and clinical tissue regenerationProtocols for the preparation and characterization of decellularized tissue and organ scaffolds for tissue engineering.Hsieh et al. [16]Preparation and characterization of decellularized tissue and organ scaffolds
Wound healingSupercritical carbon dioxide-decellularized porcine acellular dermal matrix combined with autologous adipose-derived stem cells: Its role in accelerated diabetic wound healing.Chou et al. [35]Accelerated diabetic wound healing
Extraction socket bone regenerationEvaluating the bone-regenerative role of the decellularized porcine bone xenograft in a canine extraction socket model.Chen et al. [36]Guided bone regeneration, extraction socket bone regeneration
Corneal replacementPreparation of acellular scaffold for corneal tissue engineering by supercritical carbon dioxide extraction technology.Huang et al. [37]Production and evaluation of biocompatibility of acellular corneal scaffold
Bone regenerationReconstruction of the orbital floor using supercritical CO2 decellularized porcine bone graft.Huang et al. [38]Orbital floor reconstruction
Corneal transplantationAcellular porcine cornea produced by supercritical carbon dioxide extraction: A potential substitute for human corneal regeneration.Liang et al. [39]Biocompatibility of acellular corneal scaffold in rabbit lamellar corneal transplantation. Potential substitute for human-donated cornea for corneal transplantation
Bone regenerationSupercritical carbon dioxide decellularized bone matrix seeded with adipose-derived mesenchymal stem cells accelerated bone regeneration.Liu et al. [40]Accelerated bone regeneration
RhinoplastyA novel 3D histotypic cartilage construct engineered by supercritical carbon dioxide decellularized porcine nasal cartilage graft and chondrocytes exhibited chondrogenic capability in vitro.Lee et al. [27]Engineered 3D histotypic cartilage construct for nasal septum reconstruction
OsteoarthritisSupercritical carbon dioxide decellularized porcine cartilage graft with PRP attenuated OA progression and regenerated articular cartilage in ACLT-induced OA rats.Wu et al. [41]Repair and regeneration articular cartilage in osteoarthritis
Osteoarthritis3D composite engineered using supercritical CO2 decellularized porcine cartilage scaffold, chondrocytes, and PRP: Role in articular cartilage regeneration.Chen et al. [30]3D composite engineered decellularized porcine cartilage scaffold in articular cartilage regeneration

Table 3.

Porcine tissues and organs had been decellularized by the SCCO2 process applied in different medical applications.

5.1 Tissues and organs

Currently, the most frequently employed decellularization technique for tissue and organ to manufacture scaffolds employing detergents are sodium dodecyl sulfate, Triton X-100, and CHAPS, branded as ionic, non-ionic, and zwitterionic detergents, respectively. Detergents were found to be effective in the decellularization of the tissues and organs, including the removal of lipids [11, 12]. Enzymes such as nucleases are also employed in limited decellularization protocols to eliminate the DNA from the tissues and organs [12, 13]. However, detergent-employed decellularization often disrupts the ECM by changing tertiary and quaternary structures of the proteins. SDS is known to proficiently eliminate glycosaminoglycans, thereby destructing the collagen structure [14]. Detergent decellularization is known to reduce the number of valuable growth factors that are vital for the recellularization of tissues. Moreover, residual surfactants and chemicals often cause cytotoxicity [15] inducing adverse effects in the recellularization of tissue and organ scaffolds (Table 3) [13, 14, 16].

5.2 Adipose tissue

Common traditional decellularization methods for adipose tissue include numerous freezing-thawing cycles, extraction of lipids with isopropanol, and enzymatic treatment. Developing a protocol for the preparation of ECM from adipose tissue in an accessible and eco-friendly manner will promote the upgrading of the methods of tissue engineering with the use of autologous material [9, 14, 17, 18, 19].

5.3 Pericardium

The existing techniques for pericardium decellularization include the treatment by non-ionic detergents such as Triton X-100, 3-3-chloroamidopropyl-dimethylammonio-1-propanesulfonate (CHAPS), ionic detergents (SDS), sodium deoxycholate, alkalis, and enzymes such as trypsin with EDTA. However, the adverse effects are commonly occurred by the above-mentioned procedures on the ECM structure and composition. The detergents such as SDS and Triton X-100 were found to denature the collagen of the ECM which was elucidated by staining fluorescently labeled collagen hybridizing peptide. CHAPS and sodium deoxycholate altered the structural organization of collagen established by the recording of the second harmonic signal and transmission electron microscopy. Decellularization of bovine pericardium tissue using Triton X-100 reduces the concentration of glycosaminoglycans by ~62–66%, and in an alkaline solution, by ~88.6%, at the initial concentration of ~0.6 mg/g [9, 14, 17, 18, 19].

5.4 Bone

The current standard method employed for bone decellularization is by high-temperature sintering at 300–1300°C. Moreover, this procedure completely removes any possible zoonotic infectious agents, in addition to the immunogenic components that existed in the animal bone tissues [20]. However, the high-temperature sintering damages the intrinsic collagen and alters the porous ECM structures of the animal bones. Bone decellularization can also be carried out by various chemical agents and techniques. The chemical process includes processing the bone with acidic and alkaline solutions and organic agents, as well as detergents and enzymes, that unavoidably alter the ECM structure. Delipidation is the key factor in decellularization processing because indisputably, the residual lipids in the bone act as a barrier to cell removal, in addition to altering its biocompatibility and osseointegration [21]. Moreover, it encourages adverse reactions which can give rise to bone resorption and encapsulate fibrosis [22, 23].

5.5 Cartilage

Decellularization of cartilage is challenging, due to its dense structure with lacunae. Generally, decellularization of cartilage is performed by the perfusion of detergents into the lacunae to break down the chondrocytes. In continuation, the detergents were washed out of the residual cellular fragments and nucleic acids. In another case, decellularization of cartilage was performed by treating with 0.05% Trypsin/EDTA for 1 day followed by 3% SDS for 2 days and 3% Triton X-100 for another 2 days [24]. Decellularization of the cartilage process includes a mixture of physical, chemical, and enzymatic steps [24]. Decellularization of cartilage by SDS and Triton X-10 resulted in only a 77% decrease in DNA content (262 ± 42 ng/mg) relative to the untreated cartilage. But, the key norms for medical devices, the decellularized tissue residual DNA content should be less than 50 ng/mg in decellularized materials. However, the dense nature of the cartilages reticular network of fibrous ECM is a substantial barrier for the detergents to penetrate. It is the key limitation of SDS and Triton X-100 in cartilage decellularization [24, 25]. Cartilage complete decellularization by SDS (2%) treatment for 4 or 8 h; however, 60% of the DNA remained in the decellularized cartilage [26, 27]. Decellularization of cartilage by using 1% SDS for 24 h and 2% Triton X-100 for 48 h preserved most of the ECM components with a complete chondrocyte’s removal. The complete decellularization of chondrocytes and the movement of seeded cells into the scaffolds during recellularization is challenging. The decellularization process in the SDS process caused the denaturation of proteins in ECM structures, which may also destroy the protein function [27, 28]. Cartilage decellularization methods such as chemical and enzymatic methods lead to disadvantages including traces of impurities and loss of ECM scaffold structure caused by the degradation of native collagen ECM structure leading to difficulty in the recellularization of the cartilage scaffold. Porcine articular cartilage decellularized by a succession of freeze-thaw cycles and 0.1–0.5% (w/v) sodium dodecyl sulfate detergent cycles with chondroitinase ABC and hyaluronidase were employed to breakdown glycosaminoglycans, resulting in the removal of 80–90% of the DNA [29, 30].

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6. Supercritical carbon dioxide extraction technology, an innovative and efficient approach for collagen biomaterial production

The conditions necessary for the decellularization processing of biomaterials frequently reject the use of traditional approaches involving destructive action on the biomaterial such as high-temperature treatment, acid, and alkali, etc. A result of the search for an alternative process leads to novel processing technologies and approaches concentrating on the direction of green technology in the first place. Supercritical carbon dioxide extraction technology comes in the first place in green technology. Supercritical carbon dioxide extraction technology owns exceptional advantages that can be employed in the production of biomaterials efficiently and cost-effectively. The most vital and important advantage of SCCO2 is the option of conducting processes at low temperatures, which offers the opportunity to work with a variety of biomaterials and thermally sensitive components such as collagen [31]. In the SCCO2 process, the low surface tension encourages the penetration of CO2 into solid and colloidal structures, which makes it competently decellularize and sterilize biomaterial and medical devices with the preservation of the structure and physicochemical properties (Table 4) [32].

Decellularization proceduresTissues and organs
Supercritical carbon dioxide extraction technologyBone, skin, cornea, cartilage, nerve, tendons, artery, pericardium, aortic-pulmonary valve, heart, liver, kidney, pancreas
Non-ionic detergents (Triton X-100)Subcutaneous adipose tissue, myocardium, pericardium, aortic-pulmonary valve, gingiva, dental pulp, bone, skin, cornea, cartilage, artery, tendon, liver, kidney, pancreas
Ionic detergents (sodium dodecyl sulphate (SDS))Muscle, tendons, adipose tissue, heart, pericardium, valves, artery, liver, kidney, testis, ovary, placentam, cornea
Zwitterionic detergents (CHAPS)Arteries, lung, heart, esophagus, cornea, nerve, liver
SonicationCartilage, kidney, artery, larynx, meniscus, trachea, aorta, nerve, cornea, osteochondral tissue, meniscus, intervertebral disc, adipose tissue, heart
High-pressure gradient systemArtery, soft tissue, heart tissue, cornea, aorta, kidney, pericardium, bone matrix
EnzymesCornea, amniotic membrane, pancreas, tracheal cartilage, atery, dermis, tendon, larynx, nerve, heart values, umblical cord artery, amniotic membrane, aorta, muscle
SolventsCornea, placenta, kidney, liver, dental pulp, heart, dermis

Table 4.

Decellularization techniques are used for tissues and organs.

In the supercritical process, the carbon dioxide gas above a critical temperature, Tc = 31.1° C, and pressure, Pc = 73.8 bar is said to be supercritical (Figure 2). In this state, carbon dioxide is neither a gas nor a liquid but possesses properties of both. The critical state of carbon dioxide is established by the phase diagram in Figure 2; varying the temperature and pressure changes the phase from solid to liquid to gas. However, at the critical point (the intersection of Tc and Pc), the difference between the liquid and gas phases disappears. The single fluid phase of carbon dioxide at this point is supposed to be “supercritical”. The decellularization of mammalian tissues was successfully carried out using the extractive properties of SCCO2 technology (Figure 3). To eliminate the immunogenicity of xenogeneic and allogeneic tissues requires decellularization. The decellularization process of the tissues to ECM scaffolds is to remove cells and antigens from the source tissue material. The ECM scaffold developed as an outcome of the decellularization process is the ECM consisting of proteins such as collagen, laminin, elastin, proteoglycans, and glycoproteins, as well as essential growth factors, angiogenesis factors [11]. Many porcine tissues and organs had been decellularized by the SCCO2 process (Figures 3 and 4) and had been applied in several different medical applications by our team as listed in Table 5.

Figure 2.

Phase diagram of CO2.

Figure 3.

Production of collagen scaffolds by SCCO2 technology.

Figure 4.

Natural collagen scaffolds prepared by SCCO2 technology.

Supercritical carbon dioxide extraction technology
PrincpleThe carbon dioxide gas above a critical temperature, Tc = 31.1° C and pressure, Pc = 73.8 bar is said to be supercritical. In this state, carbon dioxide is neither a gas nor a liquid but possess properties of both. At the critical point, the difference between the liquid and gas phases disappears. The single fluid phase of carbon dioxide at this point is supposed to be ‘supercritical’. This supercritical liquid can penetrate the tissue and organs efficiently to break down cellular components, which are washed off, with unaltered ECM scaffold.
AdvantagesGreen technology, cost-effectively, low temperatures, non-flammable, easily available, non-toxic, non-explosive, no chemical traces, no organic solvent, low viscosity, low surface tension, high density, gentle treatment, high productivity, fast and efficient, continuous and automatic with very low idle and turnaround time, non-corrosive, odorless, colorless. “Generally Regarded as Safe”.
DisadvantagesNo known disadvantages related to tissue decellularization. However, expensive equipment and the analysis process. Operated at the high pressure 1000–5000 psi.
ApplicationsOrgan snd tissue decellularization, which can be used as a “high end medical devises”.

Table 5.

Supercritical carbon dioxides principle, advantages, disadvantages and applications.

6.1 Aorta

The first effort for the decellularization of the porcine aorta employing SCCO2 with the cosolvent as absolute ethanol was reported in 2008 [42]. The structural analysis depicted that the addition of ethanol encourages the removal of cellular material such as nuclei and phospholipids, which was unattainable without the use of SCCO2 as a cosolvent. The results showed a decrease in the amount of phospholipids which depends on the time of processing, pressure, and rate of venting in the reactor. Altering the conditions, the lowest residual amount of phospholipids was 20%, which was attained as a result of 20 min at 15 MPa and 37°C. During the progression SCCO2 process with the ethanol system, the aorta obtains rigidity, which reflects upon the character of the stress-strain diagrams. It is related to the dehydration of the aorta tissue due to the hygroscopic nature of ethanol and SCCO2 dissolves up to 0.5% water [43]. The insignificant changes in mechanical properties and the deviations are not functionally significant [42]. This is the basics in the field of SCCO2 decellularization; however, this process was not continued, due to the fast progress of methods of decellularization using detergents, enzymes, and other physicochemical methods. The preparation of biomaterials using SCCO2 was resumed due to renovating the interest in solving the problems of decellularization and the factors such as the growth of new instrumentation, transition to green chemistry.

In the SCCO2 decellularization process, the native collagen scaffold remains completely intact, even the smallest of the collagen strand (Figure 5, dermis ECM) as shown in the scanning electron microscopic photos of several different porcine tissues and organs. Therefore, we believe SCCO2 decellularization is superior to other decellularization processes and thus the holy grail technology for the preparation of collagen scaffolds for tissue engineering and regenerative medicine. The process of decellularization of the aorta by SCCO2 was continued in 2017 by altering the protocol using 70% ethanol and the processing was executed for 1 h at 37°C in addition 17.2 and 31 Mpa [44]. The results of histological studies and residual DNA exhibited complete elimination of the cellular debris from the aorta tissue is accomplished at 31 MPa. However, the ECM structure of the aorta is significantly altered at higher pressure, and the organization of the layers of the aorta external and internal layers is altered. These alterations in the aorta are capable of encouraging the development of embolism and aneurism in the case of grafting, which is a severe constraint for the clinical use of the aorta graft. In addition, these alterations of the aorta structure change the mechanical properties of an ECM.

Figure 5.

Porcine bone derived products.

To treat ischemic diseases, cardiac tissues were decellularized using SCCO2with a cosolvent of absolute ethanol, leading to the formation of a hydrogel-based on an ECM, a source of glycosaminoglycans, proteins, and growth factors [13]. To attain the determined effect, the pressure was elevated to 35 MPa, and the time of the processing was extended to 6 h. The cardiac tissues were then rinsed in a solution of DNase I for 5 days. ECM components responsible for angiogenesis are preserved in the SCCO2 decellularization; however, 1% SDS altered the ECM. Upon subcutaneous implantation of the hydrogel to mice induced angiogenesis. Subsequently encouraged the development of vessels to a significantly superior extent in comparison with the SDS treated and control gel based on type I collagen. Therefore, decellularization using the SCCO2 opens up projections for the progression of bioinks for bioprinters and the formation of three-dimensional structures based on hydrogels [13].

6.2 Cornea

The porcine cornea was decellularized by SCCO2 [45], the cornea tissue was initially subjected to osmotic shock by changing 2 M NaCl solution and deionized water. The process of SCCO2 was done with the cosolvent addition of 60% ethanol at 35 MPa and 45°C for 80 min. In this process, it is likely to eliminate cellular components from the corneal tissue with the conservation of the suitable optical properties of the cornea. However, the decrease in the quantity of glycosaminoglycans and structural proteins during the processing in SCCO2 directed to the alterations of the structural organization of the corneal ECM. In the traditional procedure decellularization by Triton X-100, the effect was less noticeable. The transplantation of the SCCO2 decellularized cornea to rabbits showed regeneration of the cornea in 2 months, which confirmed the migration of keratocytes and corneal epithelial cells to the implanted cornea. In addition, no adverse rejections, inflammation, or angiopoiesis was observed in the implanted cornea. For the first time, the results of the regeneration of corneal tissues with the use of SCCO2 decellularized transplants over the long term were described. The physical decellularization method of the cornea was established previously by the destruction of cells under the action of high pressure up to 100 MPa. However, the high-pressure method involves complex and costly hardware.

The SCCO2-decellularized corneas displayed intact stromal structures and appropriate mechanical properties and had biocompatibility. Additionally, no immunological reactions and neovascularization were observed after lamellar keratoplasty in rabbits without complications. The transplanted decellularized corneas became transparent within 2 weeks of surgery. The decellularized corneas were completely re-epithelialized within 4 weeks. In conclusion, SCCO2 decellularized corneas could be an ideal and useful scaffold for corneal tissue engineering [37]. The SCCO2 technology-mediated production of the acellular porcine cornea (APC) depicted complete cells and non-collagenous protein removal relative to the Triton-sodium dodecyl sulfate decellularization process. APC presented excellent biocompatibility in rabbit lamellar corneal transplantation with a follow-up to 1 year. APC can be a potential substitute for human-donated cornea for corneal transplantation in the near future [39].

6.3 Bone

Decellularized bone tissue matrix produced by SCCO2 [46], bovine cancellous bone was treated at 35 MPa and 50°C for 30 min with 25 min in a dynamic mode at a rate of the flow of SCCO2 of 16.9 g/min and 5 min in a static mode of supercritical process. Subsequently, bovine cancellous bone was treated with a 7% solution of NaCl for 12 h first and then in a 0.1% solution of H2O2 for 48 h. On comparing lipid removal in bovine cancellous bone by SCCO2 with traditional extraction with n-hexane in a Soxhlet apparatus, the SCCO2 removed lipids 14% more efficiently. The biocompatibility of the SCCO2 decellularized bone was proved by seeding and culturing with mesenchymal stem cells. However, mechanical properties and immunogenicity of the SCCO2 decellularized bone were not determined. Similarly, xenogeneic bone decellularization [47] by SCCO2 was done by rinsing with a 3% H2O2 solution and processing in the subcritical water, and final processing in SCCO2.

The SCCO2 technology was used to produce a series of novel decellularized porcine collagen bone grafts (DPB) in an assortment of shapes and sizes (Figure 5, cancellous bone). The native intact collagen was preserved in the SCCO2 processed DPB was confirmed by Masson trichrome staining. The cytotoxicity and biocompatibility tests according to ISO10993 and their efficacy for bone regeneration in osteochondral defects in rabbits were evaluated. The rabbit pyrogen test confirmed DPB was non-toxic. In vitroand in vivobiocompatibility tests of the DPB did not show any toxic or mutagenic effects. in vitrocytotoxicity test, in vivopyrogen study, in vitromammalian cell gene mutation test, and systemic toxicity study in SD rats. The DPB produced by SCCO2 exhibited similar chemical characteristics to human bone, no toxicity, good biocompatibility, and enhanced bone regeneration in rabbits. Therefore, the potential application of the SCCO2 extraction technique to generate a native decellularized bone scaffold for regeneration in human clinical trials [23]. The DPB produced by SCCO2 on alveolar socket healing after tooth extraction had promising bone regeneration properties similar to that of Bio-Oss® in a canine tooth extraction socket model [36].

The DPB produced by SCCO2 ABCcolla® Collagen Bone Graft, was used for the reconstruction of the orbital framework in humans. The orbital defects were fixed by the implantation of the ABCcolla® Collagen Bone Graft. All subjects showed improvement of enophthalmos on computerized tomography at week 8 follow-up. No replacement of implants was needed during follow-ups. Thus, ABCcolla® Collagen Bone Graft proved to be safe and effective in the reconstruction of the orbital floor with high accessibility, high stability, good biocompatibility, low infection rate, and low complication rate [38]. The DPB produced by SCCO2 seeded with adipose-derived stem cells (ASCs) boosted callus formation in a segmental femoral defect. The mechanism of DPB might be modulation in the expression of BMP 2 and osteocalcin, thus leading to enhanced bone regeneration and new bone formation in a rat segmental femoral defect model. Thus the DPB scaffold is an excellent biomaterial for bone tissue repair. Implantation of the DPB seeded ASCs stimulated endochondral ossification for substantial bone regeneration. The DPB seeded ASCs system is of clinical relevance for segmental defect bone regeneration [40].

6.4 Acellular dermal matrix

The SCCO2 decellularized porcine acellular dermal matrix (ADM) seeded with autologous adipose-derived stem cells (ASCs) in streptozotocin (STZ)-induced diabetes mellitus rats showed the wound healing rate increased in diabetes mellitus. Diabetes mellitus wound treated with ADM-ASCs showed a significantly higher wound healing. ADM-ASC-treated rats showed significantly increased epidermal growth factor, Ki67, and prolyl 4-hydroxylase and significantly decreased CD45. The intervention comprising ADM decellularized from porcine skin by using SCCO2 and ASCs was proven to improve diabetic wound healing. The SCCO2 produced ADM-ASCs had a positive effect on epidermal regeneration, anti-inflammation, collagen production and processing, and cell proliferation; thus, it accelerated wound healing [35].

6.5 Cartilage

Cartilage tissue engineering that combines the triads of decellularized porcine cartilage graft as a scaffold, plasma rich platelet (PRP) as signal, and chondrocytes as the cell to attenuate anterior cruciate ligament transection (ACLT)-induced OA progression and regenerate the knee cartilage in rats. The SCCO2 decellularized porcine cartilage graft (dPCG) significantly reduced the ACLT-induced OA symptoms and attenuated the OA progression. The histological analysis depicted cartilage protection by dPCG. The repair and attenuation effect were proved by dPCG in the articular knee cartilage damage as evidenced by safranin-O, type II collagen, aggrecan, and SOX-9 immuno-staining. To conclude, intra-articular administration of dPCG with or without PRP is efficient in repairing the damaged cartilage in the experimental OA model [41]. A 3D composite was constructed using SCCO2-dPCG that promotes chondrogenic marker expression in vitro. The in vivoimplantation of 3D composite to cartilage defect exhibited significant regeneration by increasing the expression of Collagen type II and aggrecan. The bioengineered 3D composite by combining dPCG scaffold, chondrocytes, and PRP facilitated the chondrogenic marker expression in both in vitroand in vivomodels with accelerated cartilage regeneration. This might serve the purpose of clinical treatment of large focal articular cartilage defects in humans in the near future [30].

6.6 Nasal cartilage

A bioactive 3D histotypic SCCO2 decellularized nasal cartilage (dPNCG) construct was engineered with adipose-derived stem cells (ADSC) and chondrocytes and cultured for 21 days. The 3D histotypic constructs produced a solid mass of 3D histotypic cartilage with significant production of glycosaminoglycans. The SCCO2-dPNCG granules are bound to one another by extracellular matrix and proteoglycan, to form a 3D structure expressed chondrogenic markers such, as type II collagen, aggrecan, and SOX-9. The SCCO2-dPNCG substrate enabled the synthesis of type II collagen along with ECM to yield 3D histotypic cartilage. This engineered 3D construct might serve as a promising future candidate for cartilage tissue engineering in rhinoplasty [27].

6.7 Atelocollagen

Atelocollagen was prepared by using SCCO2 technology. To our knowledge, we are the first to use SCCO2 technology to produce atelocollagen. The sliced porcine skin was subjected to a proprietary SCCO2 for decellularization. The decellularized porcine skin scaffold was freeze-dried and freeze-milled to granules and subjected to enzymatic hydrolysis using pepsin in acidic conditions, then subjected to ultrafiltration for pepsin and telopeptide removal. The atelocollagen solution was filtered through a 0.2-μm filter for sterilization. The acidic atelocollagen solution was subjected to fibrillogenesis by bringing the pH to 7, then centrifuged to obtain the atelocollagen slurry. This slurry was then freeze-dried to obtain atelocollagen dry powder [34]. The whole process saves a lot of time and cost as compared to the traditional collagen purification process. Atelocollagen prepared by SCCO2 followed by pepsin digestion of the telo-peptides process showed complete removal of the telo-peptides as compared to the traditional purification process [34].

6.8 Skin

The SCCO2 technology was employed to decellularize porcine skin to produce a collagen matrix (Figure 6). This novel collagen matrix was developed to accelerate wound healing for hard-to-heal or delayed wound healing clinical conditions. The collagen matrix produced by SCCO2 technology from porcine skin is chemically comparable and biocompatible to human skin. The SCCO2 produced collagen matrix showed complete decellularization, the chemical content was found to be type I collagen and characteristic features were similar to that of humans. The collagen matrix proved to be non-toxic in in vitrocytotoxicity-agar diffusion test, in vivopyrogen study, in vitromammalian cell gene mutation test, acute systemic toxicity study in mice, systemic toxicity study in SD rats, intracutaneous irritation test, skin sensitization study (maximization test), and muscle implant study. In the porcine excision full-thickness skin wound healing model, the collagen matrix cocultured with fibroblast and keratinocytes exhibited decreased inflammation, complete epithelization, and enhanced wound healing [33].

Figure 6.

Porcine skin derived products.

6.9 Adipose tissue

The SCCO2 process was used for the decellularization of adipose tissue extracellular matrix [48]. The adipose tissue was subjected to the SCCO2 process for 3 h at 18 MPa and 37°C with the addition of ethanol as the cosolvent. The decellularized adipose tissue consisted of the extracellular matrix components and was free from lipids. The decellularized adipose tissue extracellular matrix can help the widespread coating progress the adhesion of cells due to the presence of active components such as collagen, laminin, elastin, fibronectin, and glycosaminoglycans. The coating of the decellularized adipose tissue extracellular matrix increases the proliferation of human endothelial cells isolated from umbilical vein, human adipose tissue-derived mesenchymal stem cells, human monocytic leukemia cells, and immortalized human keratinocytes on a plastic culture plate and does not induce the production of the proinflammatory phenotype of macrophages. The decellularized adipose tissue extracellular matrix was used as a model for the investigation of the action of anticancer drugs on cells for breast cancer, which is similar to the native condition [49]. The SCCO2 decellularization contrasts with the prevailing methods in the rapidity and cost-effective nature. Traditional methods of decellularization of adipose tissue include several freezing-thawing cycles, extraction of lipids with isopropanol, and enzymatic treatment [49, 50]. The development of the SCCO2 decellularization for the preparation of an extracellular matrix from adipose tissue is an environmentally friendly approach that will endorse the development of the methods of tissue engineering with the use of autologous material.

We produce tissue and organ scaffold using SCCO2 extraction technology, such as liver, kidney, heart, pancreas, artery, skin, bone, cartilage, and cornea [16]. Table 5 listed our works on the various tissue and organ scaffolds extracted by SCCO2 technology for tissue engineering applications [16]. The ultimate goal of TERM is to use the tissues and organs produced by SCCO2 from the porcine or bovine to regenerate the human tissues and organs (Figure 7). We hope to develop the whole animal application without any waste materials, which suits the purpose of the circular economy. Eventually, we intend to regenerate any human tissue and organ by its animal counterpart.

Figure 7.

SCCO2 decellularized biomaterials for TERM.

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7. Conclusions

Substantial progression in the field of TERM and scaffold biomaterials engineering by SCCO2 proposes extended potentials to acquire novel, effective achievements, which may be applied in biomedical applications. Recently, the interest in natural biomaterials produced by SCCO2 technology for medical devices production has increased, and a greater number of in-depth studies are done to better detect their likely applications related to chemical and physical characteristics and the extraction procedures, which do not modify their structural properties and biocompatibility. Tissue engineering approaches have become a valid alternative for body structure and function restoring, natural scaffold biomaterials produced by SCCO2 technology are also used as biomimetic scaffolds with controlled degradation rate in vivoand regeneration. In vitroand in vivostudies have shown the advantages related to natural scaffold biomaterials produced by SCCO2 technology use in the regenerative medicine field.

The SCCO2 decellularization technology as compared to other traditional processes is a minimally manipulated process and thus cost-effective, and gentle to the natural collagen scaffold ECM structure. Therefore, SCCO2 decellularized scaffolds might contain unaltered signals that are indispensable for stem cell adhesion, migration, homing, proliferation, and differentiation. No chemicals and solvents were involved in the process, therefore it is eco-friendly. It destroys bacteria and inactivates viruses during the process. SCCO2 technology costs only about 1/10th of the traditional process. Different tissues and organs from animals such as pigs, cows, horses, sheep can be used to produce decellularized scaffolds. The most important and key point is SCCO2 process drastically reduces immune rejection.

Our study indicated that the natural collagen scaffolds prepared by the SCCO2 process might be able to induce stem cell differentiation in vivo, with the help of the growth factors and cytokines in the microenvironment. The signal for stem cell differentiation could be pre-built by the combination of various genotypes of 29 collagen polypeptides during scaffold synthesis, which exhibits different signals in different tissues and organs that guide the stem cells to differentiate into the right cell types. The revelation of this intrinsic signal will be our future research focus. Before that, we boldly hypothesize that any organ decellularized by the SCCO2, with the intact scaffold structure, can be reconstructed in vivowhen implanted back into the live animal with the proper connection of blood circulation to bring in the stem cells required for the organ regeneration. We are testing this hypothesis and hope to find out soon. The application of biomaterials produced by SCCO2 technology to tissue engineering, in modern-day science is using the natural biomaterial with the most suitable performance in vivo, able to promote cell proliferation and differentiation in damaged tissue to restore the normal architecture of ECM. To conclude, TERM strategies particularly in the orthopedic and plastic surgery clinical field epitomize an effective and sophisticated alternative for the future, but their success firmly rests on an ever in-depth knowledge regarding the features of the scaffold biomaterial.

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Acknowledgments

This research was financially supported by the Southern Taiwan Science Park Bureau, (107SMIC-RC02; BX-01-03-05-108 and 108CB01), Taiwan, R.O.C.

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Written By

Periasamy Srinivasan and Dar-Jen Hsieh

Submitted: December 6th, 2021Reviewed: January 3rd, 2022Published: February 7th, 2022