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.