Bienestar

Regarding endochondral ossification, it is a very unique transition process, which includes two distinguished stages: chondrogenesis and osteogenesis. Cartilage matrix consists of highly hydrated proteoglycan embedded into a type II collagen network. On the other hand, bone tissue is more rigid than cartilage and impregnated mainly with hydroxyapatite (HA) and type I collagen. Regarding the endochondral bone tissue engineering, the scaffolds should be able to support cells to form transient cartilage‐like tissue in vitro and also promote the calcification of such cartilage template into bone after in vivo implantation. The following sections describe the cell‐scaffold systems that have been explored so far for the generation of bone tissue via endochondral ossification approach.

5.1 Calcium phosphate (CaP) ceramics

Before being used as scaffold materials, CaP ceramics have been widely applied as bone substitutes and implant coatings because of their resemblance to the mineral composition of the natural bone. Commercially available CaP‐based biomaterials include HA, β‐tricalcium phosphate (β‐TCP), and biphasic CaP (BCP, containing HA and β‐TCP). The CaP ceramics are generally considered to be bioactive and osteoconductive, which posses the ability to guide formation of the new bone tissue tightly along their surfaces. This has made them the superior scaffold candidates for bone tissue engineering. However, the development of three‐dimensional in vitro models of bone using bioceramics remains an important challenge that is still to be overcome. CaP ceramics are reactive and their reactivity depends on their characteristics (such as composition, dissolution, sintering temperature, microstructure). When ceramics are cultured in vitro, calcium and phosphates are released in the medium and, as a result, can hamper cellular function [27]_{. Since}

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contact for a certain period in vitro before being implanted, a number of studies have been focusing on solving the difficulties of removal of excessive ions, as well as oxygen and nutrients supply to cells within ceramic scaffolds [28]_{. One solution}

is using bioreactors to convey the medium directly throughout the interconnected pores to continuously introduce nutrients and remove wastes, so that the cell viability and activities can be favored in long‐term culture within 3D ceramic scaffolds [29]_.

Teixeira et al. reported that a BCP scaffold, consisting of 60% HA and 40% β‐TCP, had the potential to support the chondrocytes isolated from chick embryonic tibia to form bone via the endochondral route [15]_{. Tortelli et al. compared the}

biological responses between murine MSCs and mature osteoblasts, when seeded in HA scaffolds and subsequently implanted in immunocompromised mice. The results showed that new vascularized bone was formed through the activation of an endochondral ossification process in the MSCs‐scaffolds complex. Conversely, osteoblasts directly formed bone via an intramembranous ossification, without however any signs of vascularization [30]_{. In another study, mouse embryonic stem}

cells (ESCs) seeded onto ceramic scaffolds and cultured in osteogenic medium, failed to form bone tissue upon implantation in subcutaneous pockets of nude mice. Differently, when ESCs‐ceramic constructs were cultured in chondrogenic differentiation medium, they showed cartilage maturation toward the hypertrophic stage, calcification and ultimately bone formation in vivo [22]_{. All together, these}

studies indicated that the origin of bone cells and the ossification type are closely related to the nature and commitment of the seeded cells.

5.2 Hydrogels

Hydrogels are networks of hydrophilic, crosslinked polymer chains with a distinctive quality to absorb water in aqueous fluids, and hence to exhibit mechanical and visco‐elastic properties that closely resemble the structural three dimensional environment of natural extracellular matrix (ECM). These attributes make hydrogels appealing substrates for cells to adhere, proliferate, secrete new ECM and eventually restore the damage tissue [31]_{. Most importantly, from a clinical}

perspective, hydrogels are available ready‐to‐use grafts, can be injected into the wound site using minimally invasive techniques and allow complete filling of irregularly shaped defects, thus avoiding an open surgery procedure. In combination with stem cells, these scaffolds have been evaluated for their suitability as potential replacements for bone and cartilage. Hydrogels can be broadly classified into natural and synthetic, based on their origin [32]_{. Natural hydrogels (e.g. agarose,}

alginate, chitosan, collagen/gelatin, fibrin, hyaluronan, and silk), exhibit excellent bioactivity, as they mimic the native extracellular environment structurally, have

unique mechanical properties and are biodegradable by enzymatic or hydrolytic mechanisms. Synthetic hydrogels can be formed by crosslinking of polymer chains through physical (temperature, UV light or pH) or chemical reactions under controlled conditions. Representative synthetically derived polymers explored in combination with stem cells for bone regeneration purposes include poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA), oligo(poly(ethylene glycol) fumarate) (OPF) hydrogels, and poly(ethylene glycol)‐diacrylate (PEG‐DA) hydrogels [33‐35]_.

In regenerative medicine and tissue engineering of bone, the vast majority of studies have been focused on the formation of new bone directly from stem cells or mature osteogenic cells encapsulated within hydrogel matrices. However, more recently, stimulation of cells toward the endochondral ossification using cell‐laden hydrogel systems has shown to be not only appealing for the understanding of the regulatory mechanisms of cartilage and bone formation, but also a potential approach for bone repair.

5.2.1 Alginate

Alginate is a linear heteropolysaccharides composed of D‐mannuronic acid and L‐guluronic acid, and are derived primarily from brown algae. Due to the presence of the carboxyl groups along the polymer chain, alginates can form gels in the presence of divalent ions such as calcium ions [36]_{. Due to easy preparation under}

gentle conditions, low toxicity and readily availability, alginate hydrogels have been extensively investigated as matrices for encapsulating cells and delivering drugs. Alginate hydrogels have been shown to be suitable matrices for generation of bone tissue via endochondral ossification when associated with committed stem cells and appropriate combinations of regulatory signals (e.g. BMP‐2, VEGF, TGF‐β3) [37]_{. Chang et al. followed the progression of osteogenically induced MSCs}

along the endochondral pathway [38]_{. At 2 weeks after subcutaneous implantation,}

cell‐alginate constructs presented islands of cartilage formation and cells had the typical round morphology of the chondrocyte phenotype. By 6 weeks, endochondral ossification with trabecular bone deposition started to appear, whereas osteoblasts and osteocytes were seen in the new bone at week 8 and at week 12, respectively. Simultaneously, increasing calcification was detected over time. Similarly, in another study, hMSC were suspended in alginate beads with a chondrogenesis‐induction medium containing transforming growth factor TGF‐β3. During the first stage of culture, specific chondrogenic markers, such as collagen type II, type X and proteoglycan, were detectable, and their expression decreased over time in concert with the increase of osteogenic markers, such as osteocalcin

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5.2.2 Chitosan

Chitosan is a linear polymer of (1‐4) β‐linked d‐glucosamine residues with N‐acetyl glucosamine groups derived from chitin, a naturally occurring polysaccharide which forms the outer shell of crustaceans, insect exoskeletons, and fungal cell walls. Chitosan is completely soluble in aqueous solutions with pH lower than 5.0 and it undergoes biodegradation in vivo enzymatically by lysozyme to nontoxic products

[40]_{. Biocompatibility, biodegradability and structural similarity to natural GAGs}

make crosslinked chitosans hydrogels attractive materials for tissue engineering applications. Moreover, the feasibility of forming porous scaffolds showed to promote osteoblastic differentiation in vitro and might support angiogenesis and osteogenesis in vivo [41]_{. Oliveira et al. fabricated the chitosan sponges via freeze‐}

drying chitosan acidic solutions and further demonstrated that these chitosan sponges with adequate pore structure and mechanical properties could serve as a support for hypertrophic chondrocytes to induce endochondral ossification [42]_. 5.2.3 Collagen

Collagen represents the most abundant protein in the human body, being the major component of bone, skin, ligament, cartilage, and tendon. It also forms the structural framework for other tissues such as blood vessels. There are at least 19 different types of collagen, but the basic unit of all is a polypeptide consisting of a three‐amino‐acid sequence (glycine, proline, and hydroxyproline) forming polypeptide chains, which wrap around one another to form the left‐ handed triple helix structure [43]_{. Due to its excellent biocompatibility, enzymatic}

degradability and resemblance to the organic composition of natural bone, collagen‐based matrices find wide application in tissue engineering and have been extensively investigated in combination with cells and growth factors [44]_.

Abrahamsson et al. seeded human MSCs suspended collagen gel on a non‐woven PCL scaffold. Cultured in chondrogenic condition, cartilaginous tissue was formed by day 21, and hypertrophic mineralization was observed in the newly formed ECM at the interface with underlying scaffold by day 45 [37]_{. Collagen hydrogels}

could also be fabricated into porous sponges before cell seeding. MSCs‐collagen/ glycosaminoglycan constructs pre‐cultured in chondrogenic culture medium and subsequently implanted subcutaneously in the dorsum of nude mice showed good cell survival and progression along the endochondral ossification route, and more interesting, blood vessels formation. Conversely, the osteogenically primed scaffolds showed a mineralized matrix of poor quality, no vascularization, likely due to too much matrix deposition or a lack of release of inductive factors, and few surviving cells, as a result of absence of oxygen and nutrients [4, 20]_{. In}

another study, chondrocytes isolated from chick embryo were cultured in collagen sponges treated with retinoic acid to induce chondrocyte maturation

and extracellular matrix deposition. Biological properties and stiffness of collagen matrices supported chondrocytes attachment, proliferation and endochondral bone maturation after implantation [45]_.