Treatment of bone defects using osteogenic cell-laden hydrogels that can adapt to the architecture of the lesion might soon become a useful tool for orthopedic surgeons (21). In the present study, we explored whether BM-MSC expanded under a Good Manufacturing Practice (GMP)-compliant production process preserved their viability, identity and osteogenic potential when combined with commercial fibrin and decellularized and deantigenized bone particles sourced from tissue bank as a TEP. We found that indeed cells remained viable and preserved their osteogenic capacity in such osteogenic formulation, so it could be potentially used in the treatment of a wide range of orthopedic conditions or even as bioink in 3D bioprinting strategies (22, 23). Regarding the phenotype, although HLA-DR expression was higher than the criteria established by the International Society for Cell and Gene Therapy (ISCT) (24), it did comply with product specifications that were approved by the competent authority (14). Indeed, varying percentages of HLA-DR expression are often reported in MSC cultures, despite meeting the rest of defining criteria (25–29).
In current clinical practice, the use of BM concentrates are becoming popular in an attempt to provide stem cells to the fracture site. Although BM aspirates are typically processed in order to isolate the MNC fraction, this is not composed only of MSC but a heterogeneous population of B-cells, T-cells and monocytes, as well as rare progenitor cell types such as hematopoietic stem progenitor cells (HPCs) and endothelial progenitor cells (EPCs), it is still unclear which component or combination of components exactly determines its tissue induction activity, which can be exerted either by direct differentiation or by paracrine activity. It has been previously described that the MNC fraction from BM can promote angiogenesis (30), mediate vascular repair, produce cytoprotective growth factors and cytokines (31), and regenerate bone (32). From all the different subpopulations present in the bone marrow, MSC contribute to a very small fraction, estimated in the range from 0.001% to 0.1% of MNC (33, 34). Despite of such low occurrence, BM-MSC can be efficiently expanded ex vivo and induced to differentiate into multiple lineages when subjected to defined culture conditions (32). In the orthopedics field, the use of pure populations of MSC is thought to promote bone formation more efficiently.
In the present study we employed fibrin hydrogel as a clotting agent, which is a commercially available product for clinical use that can also be manufactured in situ on demand either as allogeneic or autologous product (35, 36). Typically, fibrin is presented in a formulation of two components: A) concentrate or purified fibrinogen, which is the precursor glycoprotein of fibrin, and B) a mixture of factor XIII, thrombin and calcium, which triggers the polymerization reaction (35). In fact, fibrin glue is a widely known product in the surgery field that is used for rapid hemostasis (37), acceleration of wound healing (38), reduction of blood loss (39), protection against bacterial infections (40) and its capacity for shaping to the architecture of the application zone (6). Since human fibrin is highly biocompatible and resorbable, it can be used as a vehicle for bioactive ingredients in tissue engineering strategies (36). Remarkably, MSC maintained their features and viability, when combined with fibrinogen. Such biocompatibility was key in the outcome observed after administration into animals, in which new tissue formation may result from either direct differentiation to osteogenic cells or paracrine activity, which is known to provide chemotactic factors both locally and systemically, or as a result of the combination of both mechanisms (11, 12, 41, 42). Our results are in the same line as those obtained by Seebach and collaborators, who demonstrated the compatibility of fibrin with MSC and obtained similar results regarding the angiogenic activity in a study inducing bone healing in rats (43).
Both fibrin gels and bony particles used as scaffolds in the present study displayed key features as a support for the repair of damaged bone tissue, providing a jelly texture that allows for the spatial localization of the cellular component with osteogenic potential, therefore adapting itself to the morphology of the fracture site. Particulated decellularized/deantigenized bone matrix from human cadaveric donors can be found commercially on different size formats and are commonly used in the clinics for bone mass augmentation. Small particles, like we employed in the present study, make possible to increase the surface area so, their natural osteoinductive and osteoconductive properties are available to cells embedded in fibrin hydrogels (5). The non-cellular components of the TEPs have the capacity to generate a biological environment that ensures the supply of nutrients to the cells and facilitates their regenerative function. Remarkably, both acellular and cell-laden osteogenic formulations showed osteogenic properties in vivo being BM-MSC-loaded TEP clearly the most potent formulation for generating extensive bone-like tissue and inducing improved vascularization. These findings are in accordance with similar studies published by Yamada and collaborators demonstrating bone regenerative activity when applying MSC-based TEP in a canine preclinical model first, and in the clinical setting afterwards (44–46). Kargozar and collaborators also reported a comparative study in rats with positive results regarding bone regeneration in the case of BM-MSC-based TEP (47). Despite of the promising outcomes of the present study, similar TEPs based in cell aggregates or cell sheets are gaining ground among the regenerative medicine field suggesting that different delivery modes of the active ingredient within the TEP may enhance tissue regeneration and therefore much effort is currently being made in order to improve such formulations (48, 49).