In recent years, the search for a substitute to cancellous bone autograft in large bone defects has focused on composite materials. Several parameters such as the nature of the conductive material, differentiation state of implanted cells, and the local supplementation in growth factors have been shown to be crucial in bone repair. Owing to the number of parameters to be tested, their relative importance and interactions still require further basic research. We focused our study on a blood-derived product as an easily available source of clinical-grade growth factors: a platelet glue obtained from cryoprecipitation of apheresis platelet products (PGCAP). We showed that, in vitro, PGCAP enhanced both proliferation and osteoblastic differentiation of bone marrow-derived MSCs. We confirmed these results by demonstrating the ability of PGCAP to improve ectopic bone formation in mice after implantation of MSC-loaded biphasic biomaterials. Interestingly, we showed that this approach was more efficient than using MSC-derived osteoprogenitor cells obtained after an in vitro osteoblastic induction of MSCs.
Several teams have worked on platelet-derived product optimization processes which led to various acronyms such as (a) PRP , (b) concentrated PRP (cPRP) , (c) 'thorn PRP' , and (d) 'Choukroun platelet-rich fibrin' (PRF) . Other studies reported that freezing/thawing procedures allowed platelet lysate (PL)  or cryoprecipitate (Cryo)  to be obtained. In our process of PGCAP preparation, platelets were activated on a glass support, leading to the initiation of coagulation without requiring exogenous factors . Then platelets were successively frozen/thawed, allowing arrest of the coagulation cascade, cryoprecipitation of plasmatic proteins (such as fibrinogen). Platelet lysis induced by the process allowed delivering platelet growth factors in the PGCAP. As shown in our results, the PGCAP preparation process allowed higher growth factor and cytokine concentrations than other tested products (PPP, PL, TC, and FCS). Moreover, Bieback and colleagues  showed that PL was more powerful to enhance MSC proliferation, while keeping their immune and differentiation potential, as compared with human serum or thrombin-activated PRP. PGCAP was obtained from apheresis products which contain 1 × 109 platelets per milliliter and this is in agreement with the platelet concentration used by Weibrich and colleagues  for bone repair. It is important to note that our cryoprecipitation process leads to a high growth factor concentration that does not alter MSC and osteoprogenitor phenotype and their in vivo capacity to form ectopic bone. Furthermore, in our process, gel formation was obtained by using endogenous fibrinogen and thrombin without the addition of several chemical molecules  or xenogenic thrombin, that could be of interest for a clinical application. Together, these results confirmed the advantage of platelet lysis combined with cryoprecipitation process to deliver growth factors in the final product as compared with the other platelet-derived products described above.
In addition to having a function in hemostasis, platelets play an important role in inflammation and tissue wound repair through paracrine pathways or cell-cell interactions . Upon activation, platelets release a wide range of factors that are involved in bone repair by promoting survival and proliferation of various cells (fibroblasts, MSCs, endothelial cells, osteoblasts, and chondrocytes) [17, 22]. Furthermore, some of these growth factors (EGF, FGF, IGF-1, and SDF-1α) have been shown to induce MSC osteoblastic differentiation  whereas others (PDGF, TGF-β, and HGF) exhibit controversial effects on osteogenesis depending or not on MSC differentiation stages [47, 48]. VEGF plays an essential role in angiogenesis , and several studies have investigated its role in the enhancement of fracture healing and non-unions . Moreover, these factors released by platelets have chemotactic effects on various cell types . Thus, all of these observations support the idea that platelet factors exhibit various effects promoting bone regeneration.
The physical and chemical properties of biomaterials are known to influence proliferation and differentiation of MSCs and osteoformation in vivo. These parameters also act on inflammatory reactions within the implantation bed. Malard and colleagues  suggested that a strong but brief inflammatory reaction associated with microparticles, which implies a massive release of cytokines, was favorable to the bone-healing process. This finding emphasized the importance of host response to tissue-engineering devices . In our study, moderate inflammation was observed but no significant differences were obtained with our various conditions. This indicates that the difference of structure between Calciresorb and Calciresorb bone like biomaterials did not seem to influence this parameter.
The geometry and macrostructural properties of biomaterials have been shown to play important roles: to supply nutrients and oxygen, to allow infiltration of cells and tissue, and to provide pores, channels, concavities, or spaces in which processes leading to heterotopic bone formation can occur without being disturbed by high body fluid refreshments or mechanical forces. In our study, confocal and histological analyses have shown a good migration of cells in pores inside biomaterial for both biomaterials (Figure 7). Higher bone formation areas were obtained with Calciresorb biomaterial, suggesting that a biomaterial combining macroporosity and microporosity is able to promote a better osteoformation. This result is in agreement with literature showing that microporosity and macroporosity are crucial for osteoconductivity [54, 55]. Moreover, the combination of HA and TCP seems to be a useful association for generating a scaffold that allows a rapid vascularization and integration during the early time point after implantation and a slow degradation [56, 57]. In our study, different results in terms of proliferation, osteogenic differentiation, and osteoformation were obtained with the two kinds of biomaterials tested. In fact, proliferation assays showed that Calciresorb bone like with 100% TCP favored proliferation. However, in a biomaterial with poor proliferative potential (BMA), PGCAP addition seemed to improve cell proliferation (Figure 4) and allowed levels similar to those obtained with Calciresorb bone like with 100% TCP to be reached. Even if BMF exhibited high proliferative potential, no difference in osteogenic marker expressions was obtained between the two biomaterials tested.
The importance of enhancing the osteogenic potential of MSCs/biomaterials has been investigated by pre-culturing the seeded cells on scaffolds in the presence of osteogenic media. Results from various studies suggest that differentiation of the cells toward osteoblast lineage and secretion of bone-like extracellular matrix may have osteoinductive properties and promote in vivo bone formation [58–60]. In our study, alkaline phosphatase expression was upregulated in osteogenic pre-induction conditions in vitro. However, the expression of other osteogenic markers was not affected (Runx2) or was slightly downregulated (osteopontin, osteonectin, osteocalcin, and collagen type 1) by this pre-induction step. Results obtained in vitro are correlated to in vivo results in which little bone formation was obtained with pre-osteogenic induction. Compared with other studies, different results have been obtained and this is certainly due to osteogenic medium composition or to the difference in the length of induction period necessary to improve bone-inductive properties of constructs [32, 61]. This observation raised the question of whether MSCs need to be fully differentiated in osteoblasts and which role they play in bone regeneration. Tortelli and colleagues  showed that the endochondral ossification process is responsible for bone formation from host origin with MSC implants but that an intramembranous ossification results from implants seeded by osteoblasts (isolated from calvaria). This group also showed that vascularization was increased in MSC implants and that bone formation was more important in osteoblast implants. Therefore, the quality and the type of ossification appear to be dependent on the origin of cells seeded on biomaterials. Other studies have confirmed this result by showing that MSCs seeded on biomaterial are able to recruit, first, host endothelial progenitors and, next, osteoprogenitor cells . MSC paracrine potential might explain these results . In fact, SDF-1 and its receptor CXCR4 are expressed by bone marrow MSCs and promote both proliferation and survival of these cells . Furthermore, transduced MSCs secreting high levels of SDF-1 displayed an enhanced ability to form in vivo ectopic bone. Another study indicates that SDF-1 secreted by MSCs induced recruitment of host progenitors and promoted survival and osteogenic capacity of MSCs . Adipose-derived stromal cells have also been shown to induce osteogenic differentiation of calvarial osteoblasts and to stimulate skeletal repair via paracrine signaling . Moreover, MSCs loaded on subcutaneously implanted β-TCP biomaterials are able to secrete a range of cytokines in the initial post-implantation phase, which correlates with an enhancement of bone formation in a later phase of implantation . This observation suggests that appropriate priming of MSCs is important to induce their secretion of specific factors at the injury site.
As Kasten and colleagues  showed, the addition of PRP increased alkaline phosphatase activity of undifferentiated MSC composites but had little effect on ectopic bone formation and only after 8 weeks. In agreement with our datas, their results showed that a pre-induction with chemical osteogenic inducers is not necessary for the promoting effect of PRP on osteogenesis. Moreover, it had been shown that osteogenic marker expressions were induced in MSCs expanded in PL without the need of osteogenic chemical inducers. In vivo, MSCs expanded in PL enhanced bone formation compared with MSCs expanded in FCS . Our results also demonstrate that PGCAP by itself is able to induce osteogenic markers (type I collagen, osteonectin, osteopontin, and Runx2) and that a combination with chemical osteogenic inducers is not favorable since it decreases expression of osteogenic markers (type I collagen, osteocalcin, osteonectin, and Runx2). Reduced expression of alkaline phosphatase was also obtained in PGCAP constructs placed in osteogenic medium, as similarly shown by Bruder and colleagues , who demonstrate that this alteration of alkaline phosphatase expression was dependent on the PDGF/ERK pathway. In vivo, we have shown again that PGCAP highly enhanced bone-like tissue formation and that the addition of osteogenic medium decreased the effect of PGCAP. In conditions with PGCAP, our result showed that cells were well distributed both in glue and in pores, inside the biomaterial before implantation. This indicates that PGCAP did not seem to hinder cell migration. In conditions with PGCAP, we consider that diffusion of nutrients in vivo was equivalent in conditions with a pre-osteogenic induction or not. Even if cells embedded in PGCAP interacted with an environment different than those present in pores inside biomaterial, the addition of PGCAP improved bone-like tissue formations in both biomaterials (Figure 8). Future experiments should be done to test the combination of PGCAP with biomaterial and MSCs in a model of large bone defect. With a subcutaneous ectopic model of bone formation, we did not explore the impact of mechanical constraints that occur in bone defect and the impact on biomaterial degradation and further bone remodeling. However, our combination should have some interest in large bone defect. Indeed, gel formation obtained from PGCAP represents an advantage in surgical procedure to treat bone defect. PGCAP also contains various chemokines, such as SDF-1, that could play a role in host progenitor cell recruitment, as demonstrated in our study by the presence of a high number of host cells on the implanted biomaterials (data not shown). Finally, PGCAP is enriched in survival growth factors, such as HGF, IGF-1, and SDF-1, that could play an important role for improving the local survival of implanted MSCs. Furthermore, we demonstrate that PGCAP acts on proliferation and differentiation of MSCs that could be of interest in large bone defect.