4 Discussion
In the present study, DP was successfully fabricated from periosteum harvested from the mini-pig cranium. In vitro experiments indicated that the prepared DP induced macrophage polarization to the pro-regenerative M2 phenotype, which subsequently facilitated the migration and differentiation of BMSCs to promote osteogenesis. Furthermore, in vivo experiments demonstrated that the prepared DP effectively promoted bone regeneration in a rat GBR model (Scheme 1). These results indicate that DP offers good potential as an osteo-immunomodulatory membrane for use in clinical GBR applications.
The goal of tissue decellularization is to remove as much immunogenic cellular components as possible while preserving the original ultrastructure and composition of the tissue ECM [14, 16, 24]. A variety of techniques can be used to decellularize tissue, including physical agents, chemical agents, and enzymes. Combinations of these techniques have been commonly used to improve the effectiveness of the decellularization process. In the present study, a promising protocol (freeze–thaw processing, Triton-X100, SDS and DNase I) was followed to prepare periosteum that was decellularized to the maximum degree while preserving the intact ECM structure as much as possible. The findings of the present investigation indicate that the prepared DP met the objective standards for decellularization, which generally include: (1) lack of visible nuclear material in tissue sections stained with DAPI or HE; (2) total quantified DNA concentration of less than 50 ng per mg dry tissue weight; and (3) DNA fragment length smaller than 200 bp. Overall, the results in this study demonstrated that the decellularization protocol applied was effective at removing cells from the periosteum tissue [15, 25].
The ideal decellularization procedure should minimize disruption of the ultrastructure and composition of ECM, which is composed of macromolecular fibers, of which type I collagen is one of the most vital structural proteins [16, 26]. Our SEM analysis indicated that the prepared DP retained an irregular fibrous surface architecture similar to the NP architecture, and the collagen integrity remained intact. Quantitative analysis of collagen content showed no significant difference between DP and NP, while relatively lower GAG loss was observed in DP mainly due to its high sensitivity to the decellularization SDS solutions. GAGs also represent an important ECM component and are thought to play a determinant role in the preservation of biological growth factors in decellularized tissues. This finding was consistent with other similar studies [15, 25]. The mechanical stress parameters were almost equivalent between DP and NP, also indicating that the collagen content and alignment were not obviously affected by the decellularization process. Furthermore, due to the increase in porosity with decellularization, the scaffold obtains better hygroscopicity, and the reduced elasticity modulus makes the membrane easier to apply in a bone defect. Overall, the prepared DP retained most of the defining composition and structure of NP.
Grafting of barrier membranes in GBR is known to result in a change in the local immune microenvironment, thereby influencing the subsequent bone regeneration. Immunity plays a key role not only in determining biocompatibility but also in modulating the activities of tissue-resident cells, hence influencing tissue regeneration outcomes. The fields of regenerative medicine and biomaterials science recently have focused on the importance of modulating the host immune response and have noted that rapid resolution of the inflammatory process is essential for tissue regeneration to occur [17, 20]. Ideally, a GBR membrane should synergistically promote both immune and progenitor cells to contribute to successful bone regeneration and avoid stimulation of a detrimental inflammatory response leading to a failure implantation [19, 21, 27]. Therefore, in the present study, we concentrated on the immune microenvironment generated by DP. Our results confirm that the DP scaffold induced macrophage polarization to the M2 phenotype, which effectively promoted the osteogenic differentiation of BMSCs and further bone regeneration.
The immune microenvironment generated by scaffold biomaterials can vary according to the different properties of the biomaterials, including their topological cues, chemistry, porosity, bioactive ion release, etc. [28-31]. Previous studies have reported that ECM derived from diverse source tissues can alter the phenotype of macrophages. For example, when grafted in cranial defects, ECM hydrogel from porcine periosteum was able to induce polarization of macrophages into the M2 phenotype [32]. Additionally, ECM of porcine small intestinal submucosa was shown to regulate a sequential M1–M2 macrophage transition to promote angiogenesis and osteogenesis both in vitro and in vivo [33]. However, porcine bone-derived ECM particles were reported to induce more M1 phenotype in periodontal defects [34]. In the present study, DP generated a relatively favorable immune microenvironment that supported bone regeneration, and we proposed that this is mainly due to the preservation of the natural ECM structure and composition. The retained original 3D periosteum structure along with biochemical cues including collagen, GAGs, glycoproteins, fibronectin and different growth factors, making this natural biomaterial more biocompetent than single-component collagen membranes.