360 ° in Making Acellular and Biocompatible Xenografts for Surgical Applications

There goes a famous saying “We can judge the heart of a man by his treatment of animals”. Now these animals help a man save his heart and vital organs through xenografts. The concept for xenograft is an explosive phenomenon in regenerative and tissue engineering. It has a wide application in many fields of medicine-Cardiology, Orthopedics, Dentistry, Gastrointestinology, Opthalmology and Dermatology. This comprehensive review presents in detail the current methods and procedures followed in the preparation of a xenograft. It helps us in the optimal selection of a suitable method the potential barriers and challenges faced during processing and clinical application with a view towards the future direction.


Introduction
Grafting is the fundamental element on which the magnanimous fields of regenerative and transplantation medicines have been laid.Grafts differ from flaps on the absence of their own blood supply.The basis of grafting is directed differentiation.The four main categories of grafts based on their origin can be-Autograft (from same person), Isograft (between genetically identical twins), Allograft (between same species), Xenograft (between two different species).
Xenotransplantation refers to any procedure that involves the transplantation implantation, or infusion in to a human recipient of either 1) live cells, tissues, or organs from a nonhuman animal source or 2) human body fluids, cells, tissues, or organs that have had ex-vivo contact with live nonhuman animal cells tissues or organs The potential targets for xenograft harvesting are-Chimpanzee, baboon, pig and cattle.Xenografts are widely used for tissue engineering applications.Of these, porcine and bovine sources have vast applications (Figure 1).They are less expensive and readily available than homografts and allografts.The porcine dermis, pulmonary artery, aorta, submucosal membrane and bovine bone, pericardium, jugular vein are popular sources of tissue harvesting.Acellular xenografts have wide application in several fields like; • Cardiology: Bioprosthetic heart valve constructed from bovine pericardial leaflets or porcine pulmonary valve are widely used in cardiac surgeries [1,2] .The valvular defects, aneurysm repair and the septal defects can be repaired with xenografts valves and pericardial patches.
• Orthopedics: Demineralized and deantigenized xenograft bone matrix can be used for orthopedic applications [3].Likewise Krackow Achilles tendon repair can be augmented with use of xenograft like porcine dermal matrix [4].
• Dentistry: Xenograft is also used in dentistry for reconstruction ISSN 2470-0991 • Gastrointestinology: Acellular xenograft has been used for hernia repair.The occurrence of infection, wound complication, gastrointestinal complication and recurrence of hernia is much lesser with xenograft mesh implants in comparison to synthetic mesh [6].
• Ophthalmology: It can also be used for complex scalp defects and eyelid defects [7].The possibility of implanting decellularized porcine cornea as a xenograft in place of a defective cornea is being explored.Bovine pericardium has fewer complications when used as a wrap for hydroxyapatite implants to enable attachment of extraocular muscles in the artificial eye [8].
• Dermatology: Porcine skin has similar composition, pain reducing traits, hemostasis function and collagen content as the human skin [9].It is used in cosmetology and burn wounds to ameliorate scar formation and infection.It can also be used to patch up an area after tumor removal, reducing chance of infection and fluid loss.In deep burns and scars, application of meshed acellular xenograft and the split-thickness skin autograft can induce tissue regeneration [9,10].The application of xenograft provides a micro-environment that initiates migration and proliferation of epithelial cells.There are many commercially available xenografts in ready-to-use form like Apligraf® (skin construct comprised of neonatal foreskin keratinocytes and a matrix made from bovine collagen and fibroblasts), Integra® (bilaminate dermal substitute composed of an outer silicone layer and an inner layer made of cross-linked bovine collagenglycosaminoglycan matrix) [11,12].
• Furthermore, use of Fetal Bovine Acellular Dermal Xenograft with tissue expansion has been currently implicated for Staged Breast Reconstruction (Pectoral extender), oncological hypopharyngeal defects reconstruction and interposition grafting in urological studies [13,14].
However pretreatment and intensive processing is necessary to remove antigenic factors, to reduce the rate of tissue rejection and inflammatory response triggered by the heterograft.The review deals with the processing of the xenograft starting from the collection of sample, decellularization, fixation and detoxification making it fit for clinical application.

Tissue harvesting and transportation
The animals selected for tissue harvesting are pre screened for zoonotic diseases and other risk factors.Desired tissue is collected under strict asepsis.The primary factor determining the success of this harvesting depends on the transport media.An ideal transport media should fulfill the following needs-maintain the viability and pH of cells, nourishment, inhibit microbial growth.However the choice of media mainly depends on the nature of tissue and the time of transportation.Some of the widely used media in various time-line are listed below for optimal selection.Jee et al. [15] used Hanks' Balanced Salt Solution (HBSS) to store bovine pericardium at 4°C along with antibiotic cocktail of Cifran, Gentamycin, Streptomycin, Cephalosporin, Amphotericin B. Balasundari et al. [16] used HBSS with cocktail of antibiotics like Cefuroxime, Gentamycin, Ciprofloxacin, Vancomycin, Amphotericin to transport the tissues at 4°C.Phosphate buffered saline (PBS) with antibiotics like penicillin and streptomycin has been used to transport porcine aortic valve conduits [17].
Roswell Park Memorial Institute medium [RPMI] can also be used as a transport media for the graft [23].
Golan et al. [24] used ice-cold RPMI to transport bovine fetal and human intestinal tissue.
Histocon is used as preservative-transport medium at 0-4°C.It is a cryoprotective and membrane-stabilizing agent [25].
Thomas et al. [26] had studied the efficacy of filtered coconut water as a transport medium, for avulsed tooth.It is sterile, containing a varied cocktail of sugars, amino acids, minerals, electrolytes.The preserving efficacy was compared with HBSS and milk.Results denote that coconut water is a better transport media compared to HBSS when storage is for more than 15 min and milk was inferior to HBSS [27].
The aim should be to deliver the tissue at the lab within 48 hours.The choice of media and additives added can be modified based on individual needs.However normal saline with antibiotics can be used if the transport time is less than two hours.

Decellularization
Decellularization is the initial step in preparing a xenograft.It involves removal of cells and debris present in the native tissue that might create an imbalance in calcium transport, increasing the intracellular calcium concentration.Which triggers Ca 2 + ions binding to cell membrane phospholipids and form calcium phosphate crystals.Thus tissue decellularization reduces in-vivo calcification a critical phenomenon in surgical application [28].Furthermore, it also removes the cellular antigenic components responsible for immunorejection specifically α-Gal and non-α-Gal T antigens [29].
Decellularization of graft tissues are generally performed using Sodium Dodecyl Sulphate, Triton X 100, Sodium Deoxycholate, Nuclease and Trypsin.However they are harsh to the tissues and even trace quantity remains in the tissue even after repeated wash.It has been reported that, these the trace quantity of detergens potentially impair the efficacy of the xenograft in surgical implantation [30].Furthermore, Trypsin has been deduced by Yu et al. [17] and Zou et al. [31] to be harsh for the tissue.Repeated quantification of decellularizing agents is a critical step in processing Xenograft.Optimal selection of a suitable method is also vital in this process.
Successful decellularization should maintain elasticity of extracellular matrix [ECM] similar to original tissue and not cause structural or architectural changes in the graft tissue and should be reduced cytotoxic in nature.To achieve this various protocols have been adopted, they are listed briefly in table 1 [8,17,21,28,29,[31][32][33][34][35], which aid in selection of suitable method for processing.

Cross-linking
Grafts are cross-linked to enhance mechanical stability.Interestingly, fixation also reduces immunogenicity of the tissue as it cross-links and masks the antigenic factors [36].In relation to this here we details different cross linking agents current used in the processing and their advantages and disadvantages.Their relative benefits typically aid in selection better cross-linker Glutaraldehyde: Glutaraldehyde is a dialdehyde that reacts with the amino group of lysine and hydroxylysine moieties to form cross links [37].It is the most commonly used cross-linking agent that is stable and most effective to fix tissues.In addition, it also acts as a sterilizing agent against bacteria, virus and fungi.However, glutaraldehyde has several shortcomings as discussed in table 2 [15,[18][19][20][21]29,32,34,[38][39][40][41][42][43][44][45][46][47].It is unable to stabilize all components of extracellular matrix of tissues, especially elastin and glycosaminoglycans [GAGs] [48].Moreover, the residual unbound aldehyde groups can trap host plasma calcium contributing to tissue calcification [49].The inflammatory response initiates activation of macrophages, which in turn triggers expression of matrix metalloprotein-9 [MMP-9] and tenascin-C [TN-C].TN-C elevates alkaline phosphatase expression leading to calcification of heart valves [37,49].In spite of these drawbacks, it is preferred due to long-term tissue stabilization and properties of preserving and sterilizing graft tissue.
Genipin: It is a naturally occurring cross-linker that is derived from geniposide, a compound isolated from fruit Gardenia jasminoides.It is less cytotoxic than glutaraldehyde.Genipin reacts spontaneously with amino acids or proteins to form dark blue pigments, thereby the aesthetic appearance is compromised [32].

1-ethyl-3-[3-dimethylaminopropyl] carbodiimide [EDC]:
EDC catalyses the reaction of carboxylic acid with amine groups to form amide bonds via an O-acylisourea intermediate.The addition of N-hydroxysuccinimide [NHS] to EDC increases cross-linking efficiency [39].Storage of the cross linked tissues in 25 mmol/L EDC reduced calcification in comparison to storage in glutaraldehyde [37].Epoxide crosslinker: Alkyl polyepoxides have been used to crosslink tissues.However tissue penetration is low as the water solubility is less and require organic solvents.Polar polyepoxide compounds like triglycidyl amine are water soluble and should therefore penetrate tissues easily and be biocompatible [46].
Neomycin Sulphate: Cross linking of grafts with glutaraldehyde compromises the architecture of GAG and elastin, as it stabilizes only the collagen component.These components are lost during processing or storage of the tissue.To preserve the GAG content, Raghavan et al. [50] devised a protocol using Neomycin sulphate, a hyaluronidase inhibitor, preventing enzyme-mediated GAG degradation.
Dye mediated photooxidation: It does not require any harsh chemicals.In the presence of a suitable photosensitizer [Methylene blue, methylene green and rose Bengal dyes, certain amino acids can be oxidized by irradiation with visible light.Therefore, dye-mediated photooxidation treated grafts holds promise as a long term implantable biomaterial [47].
Several methods of cross-linking have been explored to overcome the drawback of glutaraldehyde.In certain studies glutaraldehyde was replaced with silk fibroin and chitosan (1:1) cross-linkage with irradiation (electron beam).It reduces the risk of calcification and cytotoxicity.However it continues to be the most widely used agent to cross-link the tissues.It is highly recommended to develop a suitable method without using harsh chemicals.The details of cross-linking agents are detailed in table 2.

Detoxification
Formation of calcium-containing mineral deposits could result in cusp or vessel stiffness, loss of pliability, and blockage of the valve and could be attributed in part, to the toxic effect of cross-linkers [51].In addition, the phosphorus group in phospholipids can bind to Calcium in extracellular fluid and initiate calcium phosphate crystal formation.

Neutralization of residual aldehyde groups:
Pre-treatment of tissues with glutaraldehyde releases residual aldehyde groups due to polymerization.These residual aldehyde moieties enhance rate calcification of the xenograft and are cytotoxic [18,29].Compounds like α-amino oleic acid is highly hydrophobic and caps the residual aldehyde groups in glutaraldehyde-cross linked tissues, reducing calcification by ~20% compared to glutaraldehyde treatment.AOA also acts as a detergent, extracting phospholipids reducing calcification sites in the cell membranes [37].Commercial valves like Medtronic (Minneapolis, MN, USA) employ α-amino oleic acid as an anti-calcification agent for its valve (AOA®).Heparin detoxicates the aldehyde groups of glutaraldehyde [52].
No react® glutaraldehyde of Shelhigh Inc., is a heparin based detoxification method, wherein the glutaraldehyde-treated grafts are impregnated with surfactants [53].However heparin demonstrated deleterious effects [54] and thus can be avoided.Sodium bisulfite, being a reductive agent, forms α-hydroxyl sodium sulfonate on reaction with aldehyde [55].Citric acid, an organic acid with four hydroxyl groups, salifies amino groups and neutralizes free aldehyde group [56,57].It also interacts with Schiff base entities generated on aldehyde-amino group reaction inducing hydrophilicity on surface of the collagen fiber.It was also deduced that citric acid detoxification improved endothelial progenitor cell adhesion and proliferation [29].
Various amino acids have been used to detoxify the residual aldehyde remaining after glutaraldehyde treatment.This could be due to the reaction between the amine groups of amino acid and the free aldehyde groups.The treatment conditions such as concentration, pH and reaction time influences the efficiency of anti-calcification [29].Amino compounds when used alone as anti-calcificant displayed calcification in tissue, but when used in combination with organic solvents decreased calcification to low amounts [58].
GAGs and non-collagenous proteins seem to be involved mainly in the regulation of calcification processes.Hyaluronic acid (HA), a GAG, is a component of the ECM.It is biodegradable, non-toxic and is not immunogenic.The anti-calcification property of GAG was confirmed of which HA reacts with the free aldehyde groups to form stable hydrazone bond.HA can also be used as a hydrogel on pericardium, by cross-linking the hydrazide bonds for a continuous anti-calcification effect [41,59].In addition, space is created when the tissues are decellularized, which increases the possibility of calcium being deposited.This can be resolved by treating the tissues with space fillers before fixation [21].The space fillers can be used synergistically with other anti-calcification techniques for a more positive effect.
Lyophilization is a freeze drying technique to store samples like tissues, preserving their architecture and viability.This process reduces the amount of residual aldehyde.Lyophilization is a favorable means of preservation of pericardium [60].

Phospholipid removal:
The presence of phosphorus as phospholipids, in the cell membrane can lead to calcification.The calcium ions from plasma, reacts with phosphorus to form calcium phosphate deposits [28].Hence the removal of phospholipids could reduce the incidence of calcification.Alcohol can reduce calcification by dissolving the cell membrane and disorder the acyl chains of phospholipids in the graft tissue [29].Ethanol is used as anti-calcificant by St. Jude Medical, Minneapolis, MN, USA (Epic® valves), and along with Tween-80, it is used by Edwards Life sciences Corporation, Santa Ana, CA, USA for its product XenoLogiX® Long chain alcohol (LCA) has structure resembling the phospholipids.LCA would replace the phospholipids and decreases chance of calcification [60].Short chain alcohol (SCA) used in combination with LCA would improve solubility in aqueous solutions, reduce micelle formation, and improve penetration into thick tissues [36].

GA crosslinked Bovine Pericardium
A combination of the three methods mitigated free aldehyde concentration.94.1% endothelial cell survival in these grafts [56] 17 Lyophilization Acellular EDC crosslinked Bovine Pericardium reduces free aldehyde content [30] Bovine pericardium Stiffness of valve leaflets increased.Opening and closing of valve altered, increase in gradient of conduit compared to untreated graft [69] GA fixed bovine pericardium Decreased inflammatory response after six months of implantation, did not increase chance of thrombus formation [70] GA fixed bovine pericardium Decreases cytotoxicity, mechanical property maintained.
[ Tannic acid [TA] is a hydrolysable polyphenol.Glutaraldehyde fixation leads to elastin degradation, decreasing the mechanical stability.TA forms multiple bonds with elastin and collagen.Preservation of elastin content inhibits elastin-oriented calcification of glutaraldehyde-treated tissue [46].

Crystallization inhibitors:
Crystallization inhibitors prevent development of calcium crystals by binding to the crystal nucleus.Pyrophosphate, a naturally occurring polyphosphate present in serum and urine was used to prevent calcification by binding to hydroxyapatite from the 1960's [61].However pyrophosphate inhibits calcification only when injected, as it hydrolyzes on oral administration and is inactivated.Hence bisphosphonate, a stable analogue of pyrophosphate and a calcium-binding compound was synthesized [61].It is known as crystal poison as it binds to hydroxyapatite crystals and inhibits further crystal growth [59].Ethylenediaminetetraacetic acid (EDTA) is a chelating agent which can be used to minimize calcification by sequestering metal ions like Ca 2+ and reducing the dimensions of the crystal.EDTA also inhibits matrix metalloproteinase.Nevertheless it was deduced that none of these methods prevent the process of crystal formation, but only minimizes it.

Novel approach: incorporating endothelial cells and/or nanotechnology
The novel approaches that may help improve this field to serve the human race can be • Incorporation of nanoparticles to increase the efficacy and aid in the healing of chronic wounds and ulcers.
• Addition of anti coagulants in valve xenografts with sustained/ controlled release.
• Addition of anti cancer drugs in sites of tumour removal seeding of endothelial cells and collagen in the decellularised tissue can improve the durability and bio compatibility of xenografts.These tissues are found to be non-cytotoxic, non-immunogenic, noncalcific and had an improved resistance to thrombogenicity [74].This approach would decrease the complications arising from compatibility issues and can be applied to generate allogenic/autogenic grafts by culturing the endothelial cells on biodegradable polymers [75].
• Coating xenografts with immune suppressive agents may help to prevent rejection.Clinical research into these methods is necessary for clinical and medical application.

Barriers in xenotransplantation
a) Immunological barriers: Humans have Xenoregulated Natural Antibodies that lead to hyperacute rejections when they receive xenograft from discordant species.This rejection is mounted on the basis that humans lack α-1,3 galactosyl transferase enzyme.The target for most human non-Gal xenoantibodies is the sialic acid N-glycolylneuraminic acid (Neu5Gc) synthesised by the CMAH (cytidine monophospho-Nacetylneuraminic acid hydroxylase) gene, which is inactive in humans [76].It can be overcome by use of genetically engineered knockout transgenic animals.The risk of transplant rejection can be estimated by measuring the panel reactive antibody levels.Cases of dysregulated coagulation episodes have also been reported.Insertional mutagenesis is a serious entity that needs to be explored in depth.b) Biological barriers: Humans are posed to the risk of xenozoonosis following transplant.The porcine endogenous retroviruses (PERV), Swine influenza virus, Japanese Encephalitis virus, Hepatitis E virus, Nipah virus, Bubaline herpes virus, Capripoxvirus and bovine arbovius pose a serious threat of viral xenozoonosis.Furthermore, transmissions of prion diseases such as Cruezfeld Jacobsons disease, bacterial and nematode infections have also been a potential barrier [77].Harvesting organs from Specific pathogen free animals may control the issues of xenozoonosis.

Conclusion
This detailed review illustrates the step wise methods in making a xenograft, the challenges met with and possible methods to overcome them.The mechanical strength of the processed xenograft is equivalent to or higher than the homograft.The anti-calcification treatment of the graft reduces the calcifying potential of the xenograft to levels comparable to homograft.Various detoxification protocols neutralize the toxic residues that remain after the chemical treatment, thus making it safe for human use.The barriers need to be taken into mind and dealt with accordingly.The prospects of making a xenograft more compatible and less toxic can be explored further, to make it an ideal one.Slaughtering of animals for graft purpose can be minimized by the use of other alternates such as homografts, prosthetic and biodegradable synthetic grafts.

Table 1 :
Detergents and chemicals involved in decellularization.
Complete decellularization, no elastic fiber distortion, configuration of collagen type I and type IV maintained, fibronectin and laminin structure unaffected, GAG content retained.RT: Room Temperature; Td: Thermal denaturation temperature; EDTA: Ethylenediaminetetraacetic acid