*4.1. Bone Tissue*

Bones are the main components of the skeletal system. They give support to the body, allow movement, and produce red and white cells in their marrow. Bone is a connective tissue and its ECM is formed by di fferent key proteins that confer tensile strength, such as collagen type I, noncollagenous glycoproteins, and proteoglycans. Calcium hydroxyapatite (HA) in the ECM serves to store minerals and provides specific properties to bone tissue, such as resistance and hardness [68], while a series of signaling molecules, such as bone morphogenetic proteins (BMPs), are also part of a bone's ECM compartment.

The properties of bone's ECM as regenerative material have been thoroughly described in the literature, and that is why diseased and damaged bones are frequently approached using bone grafts. Bone autografts are the best option in order to avoid rejection, while allografts and xenografts are often used [46,69–72]. Implantable ceramic sca ffolds are also frequent in clinical settings as osteoconductive materials, and some of these products are bovine or porcine bone HA calcareous matrices obtained after heat-treating bones in a mu ffle furnace to remove all organic compounds, including cells [68,73].

The first use of decalcified bone as bone implantable material was described as early as 1889 by Senn [74], when he used muriatic acid as a decalcification agent, followed by washing and alcohol sterilization before implantation in human bone defects. However, the experimental evidence of the demineralized bone matrix (DBM) as an osteoinductive material was established by Urist in 1965 [52]. At present, osteoinductive activity in the bone matrix is largely related to some of the BMPs present in bone's ECM, and therefore, it is known that demineralization processing has to be done with the aim to preserve BMPs' biological activity. In any case, the DBM obtained by di fferent decalcification methods is commercially available and extensively reported in clinical settings as an osteoinductive implantable material suitable for treating bone defects [70,75,76]. Hydrochloric acid (HCl) and EDTA are common decalcifying agents, while chloroform and methanol can be used for lipid extraction. Then, the DBM can be snap frozen, lyophilized, or kept at −20 ◦C until necessary [71,77].

The decellularized bone matrix (DecBM) is frequently achieved by chemical methods, such as EDTA in combination with trypsin or SDS, along with ammonium hydroxide [46,70]. Alternatively, thermal shock can be used, together with Triton X-100, for e ffective osseous tissue decellularization [44]. Additionally, high-hydrostatic pressurization, a physical method, has been used with good results regarding bone decellularization [60]. Nucleases and dehydrated alcohol are used as complementary and final steps in order to remove waste nuclear acids and other cellular remains [53,54].

Some authors have described specifically the serial decalcification and decellularization steps in their protocols toward the generation of cell-free demineralized implantable materials [71]. In this sense, hydrogels made from the ECM of decalcified and decellularized bone are quite common due to its versatility and osteoconductivity [53,70].
