Combined Application of BMP-2 and Naturally Occurring Bioactive Factor Mixtures for the Optimized Therapy of Segmental Bone Defects

Critical bone defects, which are mostly caused by traumatic, infection- or tumor-related segmental bone loss, represent a regenerative therapeutic problem that has not been solved yet [1]. In addition, bone regeneration can be negatively influenced by unfavorable metabolic conditions such as osteoporosis, diabetes mellitus or infections. Several bone-specific growth factors have been used in an attempt to improve bone regeneration but few have made it to clinical applications [2]. Recombinant growth factors, such as recombinant human bone morphogenetic protein-2 (rhBMP-2), which is used clinically at high local doses (up to 1.5 mg/cm3), can lead to serious side effects such as paralysis due to ectopic bone formation that damages the spinal cord and nerves [3], [4], [5]. Effective bone graft substitutes are required to immobilize bioactive substances in order to enable a local, time-delayed release in the defect area.

The success of the gold standard for bone regeneration, that is autologous bone transplantation, is based on the presence of both natural growth factors and cells with regenerative potential. However, due to problems like limited availability and complications resulting from donor site morbidity, it is important to develop alternative bone substitutes. A promising strategy is to apply the principles of in situ tissue engineering to promote the ingrowth of osteogenic cells and the formation of a vascular network within the material [6], [7], [8]. By functionalizing porous bone graft substitutes with signaling factors, cells from the surrounding host tissue are attracted and stimulated, thus addressing the problem of avital areas inside the scaffolds [9,10].

The combination of different bioactive factors has been shown to be more effective for the complex process of bone regeneration than the application of single factors and consequently a more advantageous side effect profile can be achieved [11], [12], [13]. Additionally, by using synergistic effects of different factors, the doses of the single factors can be reduced. Instead of using several recombinant growth factors that are quite expensive, different natural sources of bioactive factors have been investigated. Beside platelet-rich plasma (PRP) as a potential source of these bioactive factors, the secretome of e. g. mesenchymal or multipotent progenitor cells as well as exosomes obtained from conditioned media were studied [14], [15], [16], [17], [18], [19]. Vascularization and angiogenesis were shown to be stimulated by conditioned media from cells that were cultured under hypoxic conditions (hypoxia conditioned media - HCM) and by extracts made of adipose tissue (adipose tissue extract – ATE) [20], [21], [22], [23].

The comparability and reproducibility of these bioactive factor mixtures are key factors, since the secretome depends not only on the cell source but also on the culture conditions and differs inter-individually. On the other hand, taking advantage of these individual differences, autologous production of bioactive factors is also conceivable.

In our recent work, we compared PRP-derived human platelet lysate (PL), conditioned medium from hypoxia-treated human mesenchymal stem cells (HCM) and human adipose tissue extract (ATE) as potential bioactive factor mixtures in in vitro experiments. We observed significant chemoattractive effects of PL and HCM on human bone marrow-derived mesenchymal stromal cells (hBM-MSC) as well as stimulatory effects of HCM and ATE on osteogenic differentiation of hBM-MSC and endothelial tube formation in an angiogenesis assay [24]. In the next step, heparin-modified scaffolds of mineralized collagen type I could be successfully functionalized with these different bioactive factor mixtures promoting cell migration and vascularization [25].

Based on these promising in vitro results, the aim of the present study was to evaluate the bone regenerative potential of these functionalized scaffolds for a possible clinical application in vivo in a murine segmental bone defect model [26]. In addition, naturally occurring bioactive factor mixtures were combined with rhBMP-2 in order to investigate if the locally required rhBMP-2 dose can be reduced and if negative side effects associated with the sole application of rhBMP-2 can be avoided. Therefore, the in vivo study was performed in 2 stages with stage 1 investigating the effects of the different bioactive factor mixtures and with stage 2 investigation the most promising bioactive factor mixtures of stage 1 in combination with rhBMP-2.

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