*3.3. Sterilization Methods*

The dECM is commonly used in in vivo implantable approaches. Therefore, in order to prevent the transmission of pathogens, the dECM needs to be sterilized to eliminate any microorganisms and to prevent infections [60]. There are some useful physical and chemical dECM sterilization techniques, although their suitability depends on multiple factors, which may need to be considered in each specific approach; for example, humidity, time of exposure, temperature, and the nature/load of the bioburden are some of the factors to be taken into account.

Among the physical methods, two groups are di fferentiated—those using heat and those using radiation. Heat techniques have very limited use in a dECM context, as dECM products are usually thermosensitive and high temperatures may denaturalize important proteins, as well as disrupt the structure [61,62]. Irradiation e ffectively eliminates microorganisms, and UV-irradiation is a common sterilization method used in research settings, mainly due to easy accessibility in terms of research labs and cell culture facilities [63]. On the other hand, gamma-irradiation is the preferred sterilization method for many pharmaceutical and clinical products, due to its high penetration, good assurance of sterilization, and feasible temperature during the sterilization process. Conversely, radiation may a ffect structural proteins such as collagen, reducing the strength of the treated material [64–66]. Therefore, gamma-irradiation is often selected as the sterilization method for dECM products, but the irradiation dosage has to be optimized for each specific case, and the properties must be assessed after treatment.

Liquid chemical wash can be used as a sterilization method. Alcohol-based sterilization is common in laboratories, as it is cheap and easy to access. This method is more e ffective at killing microbes in aqueous solutions, but may also a ffect the protein structure in dECM pieces. Phenols act by disrupting membranes, precipitating proteins, and inactivating enzymes. They are bactericidal, fungicidal, and mycobactericidal, but are ine ffective against spores and most viruses. Aldehydes are alkylating agents that damage nucleic acids and kill all microorganisms, including spores. In contrast to alcohols, which are volatile, phenols and aldehydes are generally toxic, corrosive, and/or irritating. Liquid chemicals must be removed after the sterilization process due to their potential toxicity in further in vivo uses [60].

Chemical methods are also useful to sterilize dECM materials, and are an alternative to physical methods. Ozone and hydrogen peroxide are traditional sterilization gases, but ethylene oxide is more commonly used in a dECM context, as the ultrastructure and the mechanical properties of the dECM are usually not altered under such treatment [64,67]. After the sterilization process, it is important to ensure the elimination of residual sterilizing agents and other possible volatile residues.

#### **4. Decellularization Methods by Tissue**

In order to choose a decellularization method, the tissue itself and the strategy to be followed have to be taken into consideration. Since each tissue has a di fferent structure and composition, the decellularization methods have to be specifically selected and empirically tested. Moreover, if the aim is to keep the tissue structure as intact as possible, chemical methods have to be chosen carefully because of the damage that they may cause to the structural macromolecules of the matrix. Therefore, the literature shows trends in the use of specific combinatorial physical and chemical approaches for each specific tissue and application, as follows.
