INTRODUCTION Bone presents continuous remodeling and a generous regenerative capacity compared to other tissues. Lifelong bone remodeling is responsible for skeletal development, responses to mechanical stimuli, and maintaining mineral homeostasis. Intrinsic regeneration capacity guarantees bone integrity as an injury repair process. Moderate-sized bone defects repair without the need for a graft. However, complex clinical conditions are requiring a considerable amount of bone, in which natural bone regeneration capacity is not sufficient to establish functional tissue recovery. In the case of significant bone defects created by trauma, infection, skeletal disorders, or in the treatment of tumor excision, the regeneration process becomes compromised [1].Strategies may be necessary to guide or accelerate the process, increasing bone quantity or quality [2]. There is intense research in bone bioengineering, seeking to overcome the limitations observed in non-healing defects and alternative methods to autologous bone grafts, in order to produce bone substitutes with the same properties of this gold standard [3]. Osteoconduction is the process of perivascular tissue, precursor, and osteoprogenitor cell ingrowth, from the bony bed into implanted frameworks. Osteoinduction is the induction of undifferentiated mesenchymal stem cells into osteoprogenitor cells, also at ectopic sites [4]. The combination of three-dimensional biocompatible frameworks with cells and growth factors may stimulate osteogenesis and osteoinduction, enhance the osteogenic capacity of transplanted and endogenous cells and thus, ensure better healing [5].Calvarial “non-healing” defects are useful in the preclinical study of bone repair to analyze strategies of tissue engineering in intramembranous bone formation. Some advantages of this defect are the orthotopic non-load bearing site, its reproducibility, mechanical stability, and the limited baseline healing plateau, above which the effect of cell/scaffold implant on osteogenesis is noticeable [6-9]. The application of membranes is indicated for Guided Bone Regeneration (GBR), to isolate the bone defect from other tissues, and for bone reconstruction [10]. Commercially available collagen membranes are widely used in in vivo studies, associated with ceramic implant material [11], demineralized bone matrix [3], growth factors [12], and cells [13]. The membranes covering bone defects in calvaria provided stability for ceramic tricalcium phosphate (TCP) inserted into the defect, resulting in higher bone amount and better mechanical properties [11]. The association of ceramic and membrane with bone marrow mesenchymal stem cells have been demonstrated to favor earlier bone deposition [13]. Calvarial “non-healing” defects are effective in the preclinical study of bone repair to analyze strategies of tissue engineering in intramembranous bone formation. Some advantages of this defect is the orthotopic non-load bearing site, its reproducibility, mechanical stability, and the limited baseline healing plateau, above which the effect of cell/scaffold implant on osteogenesis is noticeable [6-10].Commercially available collagen membranes are widely used in in vivo studies, associated with ceramic implant material [11], demineralized bone matrix [3], growth factors [12] and cells [13]. The membranes covering bone defects in calvaria provided stability for TCP inserted into the defect, resulting in higher bone amount and better mechanical properties [11]. The association of ceramic and membrane with bone marrow mesenchymal stem cells has been demonstrated to favor earlier bone deposition [13].Tissues that are mainly available in maternity hospitals, and often discarded, can be a reliable source of allogenous cells and collagenous matrix for bone tissue engineering applications. Importantly, umbilical cord mesenchymal stem cells have been shown to stimulate vascularization and bone formation in vivo [14, 15]. In addition, amniotic membranes are known to be a source of stem cells and collagenous scaffold [16, 17]. Mesenchymal stem cells from amniotic membranes can stimulate osteogenic and angiogenic differentiation of various cell sources, including, adipose-derived stem cells [18]. Moreover, amniotic membranes present great potential in clinical application [19], tissue engineering [17], and the removal of the epithelial cell layer minimizes the risks of adverse immunological responses [20, 21].Amniotic membrane association with stem cells from bone marrow [22] and adipose tissue [23, 24] demonstrated that Decellularized Human Amniotic Membrane (DAM) is an excellent cell-carrier in tissue regeneration applications. Bone and periodontal tissue engineering studies have demonstrated that DAM can provide a preferential environment for osteogenic differentiation of dental apical papilla cells, increase the expression of osteogenic marker genes, and deposition of mineralized matrix in vitro [25]. Periodontal ligament stem cells transfer onto DAM [26], and ASCs cultured on DAM [27] have been shown to stimulate periodontal regeneration in vivo . Interestingly, double-layered cell transfer technique on DAM allowed transplantation of periodontal ligaments stem cells and osteoblasts, and enhanced bone formation in calvaria when compared to single-cell type transplantation [28]. The application of amniotic membrane in calvarial defects has been found to promote more significant bone regeneration compared to defect without graft, but less than the association with ceramic material (HA) and osteoinductive factor Bone morphogenetic protein (BMP) [29]. Interestingly, DAM associated with ASCs were also used successfully in a calvarial defect in rabbits [24].Studies using multipotent ASCs, since their first reports [30, 31] have demonstrated their potential as a significant source of adult stem cells in regenerative medicine. Some significant advantages of ASCs in bone engineering compared to Bone Marrow Stem Cells are the facility in harvesting, higher cellular yield, and proliferation capacity [32]. The application of the patient autologous cells from fat, transferred in order to enrich and accelerate the bone regeneration process was reported, in cranioplasties associated with TCP [33], in maxillary defect associated with titanium, TCP, BMP [34], and graft [35].Earlier studies investigating calvarial defects without cell expansion from the adipose tissue have used various materials, including fragmented adipose tissue [36, 37], Stromal Vascular Fraction (SVF) associated with polylactide (PLA), demineralized bone matrix (DBM) [38], and hypertrophic cartilage [39]. After isolation, ASCs were investigated as perivascular cells associated with poly(lactic-co-glycolic acid) (PLGA)/ hydroxyapatite (HA) composites [9, 40], stem cells associated with PLGA [41], stem cells associated with PLGA/HA composites [40, 42-48], stem cells associated with Whitlockite [49], with acellular collagen dermal matrix [50], with HA/TCP bioceramics and hydrogel [51], silicate bioceramic [52], and duck-beak bioceramic [53].Investigations on ASCs participation in calvarial bone defect repair have reported the occurrence of significant cell migration to the lesion site after intravenous cell administration [42], and paracrine effect of ASCs on in vitro and in vivo osteoblastic cell differentiation [47]. There was a significantly higher stimulation in cell association of immediately prepared defects, compared to cell graft with established bone defects [46]. There is considerable evidence indicating that ASCs cells may contribute to periodontal regeneration [54]. Our results demonstrated enhanced in vivobone regeneration by undifferentiated adipose-derived stromal cells loaded onto a decellularized human amniotic membrane.
