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\begin{document}

\title{Decellularized periosteum promotes guided bone regeneration via
manipulation of macrophage polarization}



\author[1]{Jiayang Li}%
\author[2]{Dongming He}%
\author[2]{Longwei Hu}%
\author[2]{Siyi Li}%
\author[2]{Chenping Zhang}%
\author[2]{Xuelai Yin}%
\author[2]{Zhen Zhang}%
\affil[1]{Shanghai Stomatological Hospital}%
\affil[2]{Shanghai 9th Peoples Hospital Affiliated to Shanghai Jiaotong University School of Medicine}%


\vspace{-1em}



  
  \date{June 14, 2024}


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\selectlanguage{english}
\begin{abstract}
Periosteum has shown potential as an effective barrier membrane for
guided bone regeneration (GBR). However, if recognized as a ``foreign
body'', insertion of a barrier membrane in GBR treatment will inevitably
alter the local immune microenvironment and subsequently influence bone
regeneration. The aim of this investigation was to fabricate
decellularized periosteum (DP) and investigate its immunomodulatory
properties in GBR. DP was successfully fabricated from periosteum from
the mini-pig cranium. In vitro experiments indicated that the DP
scaffold modulated macrophage polarization toward a pro-regenerative M2
phenotype, which in turn facilitated migration and osteogenic
differentiation of bone marrow-derived mesenchymal stem cells. A rat GBR
model with a cranial critical-size defect was established, and our in
vivo experiment confirmed the beneficial effects of DP on the local
immune microenvironment and bone regeneration. Collectively, the
findings of this study indicate that the prepared DP possesses
immunomodulatory properties and represents a promising barrier membrane
for GBR procedures.%
\end{abstract}%



\sloppy


\textbf{1 Introduction}

Guided bone regeneration (GBR) is a therapeutical strategy widely
applied to promote new bone regeneration mainly in the maxillofacial
region, especially in cases of periodontal and peri-implant bone defects
{[}1{]}. As an important step in GBR procedures, a barrier membrane is
inserted between the soft tissue defect and the bone defect to prevent
ingrowth of competing epithelial and connective tissue {[}1, 2{]}.~This
creates a segregated space for slow-migrating progenitor cells and/or
stem cells with osteogenic potential to colonize the defect site and
ultimately achieve bone regeneration. Given the key role that the
barrier membrane plays in GBR procedures, researchers have continued
pursuing novel strategies to develop superior GBR membranes.

Periosteum is a thin vascularized connective tissue that covers the
external surface of bone (with the exception of articular bone) and
plays a key role in bone regeneration {[}3, 4{]}. Periosteum is known to
be a niche of progenitor cells and local growth factors, as well as to
serve as a natural scaffold for recruitment of cells and biological
factors {[}5, 6{]}. Additionally, this tissue shows prominent smart
material properties, such as direction- and flow rate-dependent
permeability {[}3, 7, 8{]}. Based on the great potential of periosteum
in bone tissue engineering applications, several attempts have been made
to apply tissue engineering methods to develop an artificial periosteum
that mimics native periosteum in structure and function {[}9, 10{]}. To
date, however, no tissue engineering strategy has been able to recreate
the unique three-dimensional (3D) microenvironment that fully
recapitulates the periosteum-specific extracellular matrix (ECM)
properties.

In recent years, in the fields of tissue engineering and regenerative
medicine, tissue-derived ECM has become an increasingly popular
biomaterial source {[}11-13{]}. It has been shown that the
decellularization process efficiently removes antigenic cellular
components while retaining the natural 3D ultrastructure complete with
the native ECM composition. Studies have demonstrated the feasibility of
preparing decellularized tissue ECM scaffolds and applied them in the
repair of multiple types of tissues and organs with varying degrees of
success {[}14-16{]}. Although tissue-derived ECM scaffolds are generally
used for the repair of non-homologous anatomic sites, site-specific
homologous tissue scaffolds have been shown to be more effective than
non-site-specific tissue scaffolds in remodeling constructive tissue.
Therefore, we speculated that decellularized periosteum, which provides
the native ECM derived from periosteum, holds great potential as a GBR
membrane. However, the related research is relatively scarce.

Based on the convergence of osteoimmunology and immunomodulation,
osteoimmunomodulation has been proposed to be an essential ability of
biomaterials for regulating bone regeneration {[}17, 18{]}. A
fundamental principle underlying this concept is that biomaterials have
immunomodulatory properties that are important for generating an
osteoimmune environment that facilitates bone regeneration. With more
detailed investigations of the interaction between host tissues and
biomaterials, the immune response is considered a necessary element in
membrane-mediated GBR procedures. Due to its ``foreign body'' nature,
the barrier membrane will inevitably alter the local immune
microenvironment and thereby influence the dynamics of bone regeneration
{[}19-21{]}. Therefore, research efforts to develop improved barrier
membranes must consider the osteoimmunomodulatory properties of the
membrane material.

In the present study, cranial periosteum of mini-pig was harvested to
fabricate decellularized periosteum (DP), which was then characterized.
In vitro, we investigated the effect of DP on the inflammatory reaction
and polarization of macrophages as well as its effect on osteogenesis.
In vivo, we examined the effectiveness of DP in GBR in rat cranium
critical defect model. Collectively, the findings of this study indicate
that the DP possesses osteoimmunomodulatory properties and represents a
promising membrane for GBR procedures.

\textbf{2 Materials and Methods}

The animal experiment protocols were approved by the Animal Experiment
Ethics Committee of Shanghai Ninth People's Hospital affiliated to
Shanghai Jiaotong University, China. The studies were designed according
to the guidelines of the Animal Research: Reporting of \emph{In Vivo}
Experiments (ARRIVE).

\emph{2.1 Fabrication of DP}

The cranium periosteum of mini-pigs was harvested using the following
procedure. Briefly, 12-month-old minipigs were anesthetized by
intraperitoneal injection of Zoletil (50 mg/kg, Virbac, France). Then
the skin and the overlying fascia of the cranium were cut to expose the
periosteum. Then the intact periosteum was carefully stripped from the
bone surface using a periosteal detacher. The harvested natural
periosteum (NP) was prepared for further evaluation or the
decellularization process.

The decellularization process for the preparation of DP involved
physical, chemical, and enzymatic methods. First a freeze-thaw process
(-80, 24 h; 37, 2 h) was repeated three times. Then the obtained
samples were treated with 2\% Triton X-100 for 12 h followed by 0.5\%
sodium dodecyl sulfate (SDS) solution for 4 h and 100 U/ml DNase I for
12 h, after which it was soaked in ultrapure water for 3 days to remove
the residual treatment reagents. Finally, the fabricated DP was stored
in sterile phosphate-buffered saline (PBS) containing 100 U/ml
penicillin and 100 \selectlanguage{greek}μ\selectlanguage{english}g/ml streptomycin for preservation.

Collagen scaffolds (COL scaffolds) for use as a control were prepared as
previously described {[}22{]}.

\emph{2.2 Evaluation of decellularization}

2.2.1 Histological and immunofluorescent evaluation

For confirmation of decellularization, DP samples were fixed in 4\%
paraformaldehyde for 24 h and embedded in paraffin. After dehydration
and paraffin embedding, the DP samples were cut into 5-\selectlanguage{greek}μ\selectlanguage{english}m-thick sections
for histological examination using hematoxylin and eosin (H\&E)
staining. The stained sections were visualized and photographed on an
optical microscope (Leica, DM3000, Germany). Residual nuclei within the
DP were observed by DAPI staining viewed on an Olympus BX53 microscope
(Olympus, Japan).

2.2.2 Quantification of DNA, glycosaminoglycan (GAG) and collagen
content

The DNA content in DP samples was determined using a Genomic DNA
Extraction Kit (Takara, China) following the manufacturer's
instructions. The GAG content in DP samples was evaluated using a DMMB
Colorimetry Kit (GenMed, USA). A hydroxyproline assay kit (Abcam, USA)
was utilized to quantify the collagen content of DP samples according to
the manufacturer's instructions.

\emph{2.3 Characterization of DP}

The surface morphology of DP scaffolds was analyzed by scanning electron
microscopy (SEM; Model: JSM-6700F, JEOL, Japan) and atomic force
microscopy (AFM; Model: Multimode \& Dimension 3100, Varian, USA).
Additionally, the scaffolds were stained with Picrosirius Red (Solarbio,
China) and viewed under a polarizing microscopy (Leica, Germany) for
observation of the collagen structure. DP samples were also
histologically examined using Masson trichrome staining and
safranin-O/fast green staining. Immunohistochemical analysis was
performed using antibody against type I collagen (dilution: 1:200,
Abcam, USA). The surface hydrophilicity was evaluated by measuring the
water contact angle with a contact angle goniometer (Dataphysics OCA 35,
Germany) using the sessile drop method. The mechanical properties of the
DP scaffolds were tested using a universal testing machine (Model: AG-IC
50KN, SHIMADZU, Japan). Dumbbell-shaped samples measuring 4 mm \selectlanguage{ngerman}× 50 mm
were prepared for mechanical testing, and the crosshead speed was set at
15 mm/min with a load capacity of 50 N. The yield stress, failure
strain, and modulus data were obtained.

\emph{2.4 In vitro experiments}

2.4.1 Cell culture

RAW264.7 cells (murine macrophage cell line) were purchased from the
Typical Culture Preservation Commission Cell Bank, Chinese Academy of
Sciences (Shanghai, China), and cultured in Dulbecco's Modified Eagle's
Medium (DMEM, Sigma-Aldrich, USA) containing 10\% fetal bovine serum
(FBS, Hyclone, USA) at 37\selectlanguage{english} in a humidified incubator with 5\%
CO\textsubscript{2}. Rat bone marrow-derived mesenchymal stem cells
(rBMSCs) were isolated, cultured, and identified utilizing a
well-established technique as previously described {[}22, 23{]}.

The COL and DP scaffolds were placed individually in 96-well plates, and
cells were seeded over the scaffolds at a density of
2x10\textsuperscript{4} cells/mL. Cells seeded on plates without
scaffolds served as controls. Cell proliferation was quantified using
the Cell Counting Kit-8 (CCK-8; Sigma, USA) on days 1, 3, and 5 after
seeding.

2.4.2 Evaluation of the effects of DP scaffolds on macrophage
polarization

Cells seeding on different scaffolds were harvested after 3 days, and
the impact of DP on macrophage polarization was evaluated by flow
cytometry (BD Accuri C6, USA) using antibodies against CD86 (1:50
dilution, BioLegend, USA) and CD206 (1:400, BioLegend, USA) along with
Dylight 488-anti-mouse secondary antibodies. The relative levels of
inducible nitric oxide synthase (iNOS) and arginase 1 (Arg-1) gene mRNA
transcripts relative to the control glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) mRNA in the different groups of RAW264.7 cells
were quantified by qRT-PCR. The primers sequences used for qRT-PCR are
presented in Table 1. For enzyme-linked immunosorbent assay (ELISA)
evaluation, the supernatants of RAW264.7 cultures were collected. The
concentrations of the cytokines interleukin (IL)-10 and TNF-\selectlanguage{greek}α \selectlanguage{english}in the
supernatant samples were quantified using ELISA kits (Dakewe
Bioengineering, China) according to the manufacturer's instructions.

For RNA-sequencing analysis, total RNA was extracted from the tissue
samples using TRIzol reagent (Invitrogen, USA), according to the
manufacturer's instructions. cDNA libraries were then constructed for
pooled RNA samples using the VAHTSTM Total RNA-Seq (H/M/R) Library Prep
Kit (VAHTSTM, China). Differentially expressed genes (DEGs) were
identified using TopHat and Cufflinks, and their expression levels were
determined by the fragments per kilobase of transcript per million
mapped reads method. The DESeq algorithm was used to screen DEGs between
groups. The biological functions of the DEGs were then investigated by
Gene Ontology (GO) enrichment analysis (http://www.geneontology.org/).

2.4.3 Evaluation of the effects of polarized macrophages on the
proliferation, migration, and differentiation of rBMSCs

To analyze the impact of macrophage polarization on the proliferation,
migration, and differentiation of BMSCs, supernatant samples were
collected from wells containing macrophages seeded on scaffolds after 3
days of culture to prepare conditioned medium (CM). BMSCs were cultured
in 96-well plates and then treated with a mixture of DMEM and CM at a
ratio of 1:1. After 1 and 3 days in culture, the CCK-8 assay was
performed to evaluate the proliferation of BMSCs.

Wound scratch assays and Transwell assays were performed to evaluate the
effects of polarized macrophages on the migration of BMSCs. In the wound
scratch assay, BMSCs were seeded in six-well plates with basal medium,
once they reached 90--100\% confluency, a scratch with the head of
200-\selectlanguage{greek}μ\selectlanguage{english}L pipette tip (Axygen, USA) was made through the cell layer. Then,
serum-free CM was added, and the cells were further incubated for 12 h
and 24 h. Cell migration was observed under a microscope (Olympus,
Japan), and the healing area was calculated using ImageJ software (NIH,
USA). Boyden chambers were used for the Transwell assay. BMSCs were
plated in the upper chamber with basal medium, and RAW264.7 cells were
seeded on different materials in the lower chambers. After culture for
24 h, the penetrating cells were fixed with 4\% paraformaldehyde,
stained with crystal violet solution, and counted under an optical
microscope (Olympus, Japan).

Alizarin Red staining (ARS) was conducted to evaluate calcium nodule
deposition by BMSCs cultured in CM with osteogenic components for 21
days. The rinsed BMSCs were fixed in 4\% paraformaldehyde and stained by
exposure to 2\% ARS solution at room temperature for 20 min. The stained
calcium deposits were dissolved by application of 10\% cetylpyridinium
chloride (Sigma-Aldrich, USA) for 15 min, and dye release was quantified
by spectrophotometry at 562 nm (Thermo Fisher Scientific, USA).

The mRNA expression levels of bone morphogenetic protein-2 (BMP2) and
runt-related transcription factor 2(RUNX2) in BMSCs cultured with CM for
3 days were measured using qRT-PCR. The relevant primers sequences are
presented in Table 1.

\emph{2.5 In vivo experiments}

2.5.1 Rat model of cranial defect

Sprague--Dawley rats (male, weighing 280--320 g) were kept in a specific
pathogen-free facility with a controlled temperature of 22\selectlanguage{ngerman}±2°C, humidity
of 55±5\%, a light/dark cycle of 12/12 h, and free access to water and
food. To create the critical-sized defect models, the rats were
anesthetized with isoflurane gas, and a skin incision was made to expose
the cranium. Then 5-mm-diameter defects were created bilaterally. Then
the defects were covered with COL membrane, DP, or left empty (blank
control group), and the wound was subsequently closed. All rats were
housed and fed routinely until being euthanized at the designated time
points.

2.5.2 Micro-computed tomography (\selectlanguage{greek}μ-\selectlanguage{english}CT) analysis

Cranial samples harvested at 8 weeks post-surgery were fixed in 4\%
paraformaldehyde. The fixed samples were scanned using \selectlanguage{greek}µ-\selectlanguage{english}CT system
(Scanco Medical, Bassersdorf, Switzerland) with 70 kV voltage, 114 mA
electric current, and 700-ms integration time. The obtained images were
analyzed with the software of the \selectlanguage{greek}μ\selectlanguage{english}-\selectlanguage{english}CT 80 system for 3D construction.
Cylinders with 5 mm diameter and 1 mm height were chosen as the volume
of interest (VOI). Bone mineral density (BMD) and bone volume/tissue
volume (BV/TV) were calculated for quantitative analysis of bone
regeneration within each VOI.

2.5.3 Histological evaluation

Immediately after \selectlanguage{greek}μ-\selectlanguage{english}CT analysis, samples were decalcified and embedded
in paraffin. Sections were prepared and subjected to Masson's trichrome
staining. ~The sections were observed under a stereoscopic~microscope
(Eclipse E600, Nikon, Tokyo, Japan), and the proportion of newly
regenerated bone was evaluated using Image-Pro Plus software (Media
Cybernetics).

2.5.4 Immunohistochemical staining

At 1 week post-surgery, collected tissues were prepared into 10-\selectlanguage{greek}μ\selectlanguage{english}m-thick
sections and stained using primary antibodies targeting the macrophage
pan marker CD68 (ab125212, Abcam, USA), M1 phenotype marker iNOS
(ab15323, Abcam, USA), and M2 phenotype marker CD206 (ab64693, Abcam,
USA). Briefly, dewaxed sections were incubated with primary antibodies
overnight and then incubated with secondary antibody for 30 min. The DAB
kit (Proteintech, USA) was used to detect immunoreactions, and stained
sections were viewed under an optical microscope (Leica DMI 6000B
Microsystems, Germany). The proportion of positive cells was calculated
under 40\selectlanguage{ngerman}× magnification.

\emph{2.6 Statistical analysis}

All quantitative data are expressed as mean ± standard deviation (SD)
values derived from independent experiments with at least triplicate
samples. GraphPad Prism v.9.0 software (La Jolla, CA, USA) was employed
for data analysis using one-way analysis of variance (ANOVA) with
Tukey's post-hoc test. P values \textless{}0.05 were considered
statistically significant.

\textbf{3 Results}

\emph{3.1 Effectiveness ofthe decellularization procedure}

Macroscopic images of NP and DP are shown in Figure 1A. HE staining of
both sample types showed a double-layered (fibrous layer and cambium
layer) structure consistent with NP as well as well-organized collagen
fibers, but cell nuclei were absent from both layers of DP. Within DP,
collagen fibers were still arranged in an orderly manner similar to that
in NP (Figure 1B). DAPI staining also confirmed that almost no cellular
components or nuclear material could be observed in DP (Figure 1C). The
DNA quantification analysis indicated that the amount of DNA in NP was
526.2±44.68 ng/mg, while that in DP was 38.2±12.39 ng/mg (Figure 1D).
The quantification of collagen content showed no significant difference
between NP and DP (115.76±11.26 and 103.20±13.26 \selectlanguage{greek}μ\selectlanguage{english}g/mg dry weight for NP
and DP, respectively, P\textgreater{}0.05; Figure 1E). The DMMB results
indicated a significant decrease in GAG content in DP (1.87\selectlanguage{ngerman}±0.07 \selectlanguage{greek}μ\selectlanguage{english}g/mg
dry weight) compared with NP (3.99\selectlanguage{ngerman}±0.07 \selectlanguage{greek}μ\selectlanguage{english}g/mg dry weight,
P\textless{}0.05; Figure 1F). In addition, only examination of DP and
not NP showed no visible bands of DNA detected by agarose gel
electrophoresis. In summary, these results indicate the effectiveness of
the decellularization procedure for removal of cellular components from
periosteum.

\emph{3.2. Physical and mechanical properties of DP}

The surface topographies of collagen, NP, and DP scaffolds were
visualized by SEM and AFM (Figure 2A\&B). As shown in the SEM images, NP
had a relatively smooth surface, while COL and DP displayed uneven and
porous surfaces with abundant collagen fibers randomly arranged in a
network pattern. Masson trichrome staining revealed the collagen fiber
structure in each group (Figure 2C). The structure and distribution of
collagen did not differ significantly between DP and NP. Picrosirius red
examination also showed that the arrangement and structure of collagen
in DP was similar to that in NP (Figure 2D). Immunohistochemical
staining of collagen I demonstrated the arrangement of type I collagen
in each group (Figure 2E), and Safranin O staining showed the presence
of identifiable GAGs in DP scaffolds after decellularization (Figure
2F). The surface hydrophilicity of each scaffold material was analyzed
by water contact angle measurement as shown in Figure 2G. Both the NP
and DP scaffolds showed good hydrophilicity. Analysis of the mechanical
properties of each scaffold type showed no significant difference in
yield stress among the groups, but that the DP scaffolds exhibited a
moderate elastic modulus (Figure 2H).

\emph{3.3 Effect of DP on macrophage polarization in vitro}

CCK-8 analysis results showed that both BMSCs and RAW264.7 cells
cultured on each scaffold material maintained similar proliferation
activities, with no significant differences found among the groups
(Figure 3A\&B). Flow cytometric evaluation indicated upregulated
expression of the M2-macrophage marker CD206 as well as downregulated
expression of the M1-macrophage marker CD86 among RAW264.7 cells in the
DP group (Figure 3C). qRT-PCR results further confirmed that the
expression of the M2 macrophage-related gene \emph{ARG-1} was
significantly higher, while that of the M1 macrophage-related
gene\emph{iNOS} was much lower in the DP group than in other two groups
(Figure 3D). Additionally, similar trends in the secretion levels of
cytokines IL-10 and TNF-\selectlanguage{greek}α \selectlanguage{english}among the groups were confirmed by ELISA
evaluation (Figure 3E).

The transcriptomes of RAW264.7 cells cultured on COL and DP scaffolds
were further analyzed. Transcription of 3092 genes in these cells was
altered after their culture on DP (\textbar{}log2fold change\textbar{}
\textgreater{}1, false discovery rate {[}FDR{]} \textless{}0.05), of
which 1405 genes were upregulated and 1687 were downregulated (Figure
3F\&G). GO enrichment analysis showed that most of the over-expressed
genes were associated with regulation of the inflammatory response,
antigen processing and presentation, cytokine activity, the major
histocompatibility complex (MHC) protein complex, as well as regulation
of angiogenesis and vasculature development (Figure 3H). Kyoto
Encyclopedia of Genes and Genomes (KEGG) analysis showed that the
over-expressed pathways were associated with inflammatory modulation,
including the p53 signaling pathway, NF-kappa B signaling pathway, and
IL-17 signaling pathway (Figure 3I).

\emph{3.4 Effect of DP-induced macrophage polarization on BMSC migration
and differentiation in vitro}

The effects of macrophages cultured on DP on the proliferation,
migration, and differentiation of BMSCs were further evaluated. CCK-8
analysis indicated no significant differences in cell proliferation
activity among the different groups (Figure 4A). Both wound scratch and
Transwell assays showed that BMSC migration was significantly promoted
by macrophages seeded on DP scaffolds in comparison to the different
control conditions (Figure 4B-E). Additionally, we observed that the CM
from macrophages cultured on DP facilitated the osteogenic
differentiation of BMSCs, as confirmed by ARS staining as well as
changes in the mRNA expression levels of \emph{Runx2} and \emph{BMP2} .
The obtained data indicate that CM from macrophages exposed to DP
promoted the migration and osteogenic differentiation of BMSCs.

\emph{3.5 Effectiveness of DP scaffold in a GBR model in vivo}

A 5-mm-diameter critical-size defect was established in a rat cranium
for our in vivo GBR model. No inflammation or infection was observed at
the defect sites in the different groups during the experimental period.
Micro-CT was applied to evaluate bone regeneration within the defects in
each group. Representative 3D reconstructed images are displayed in
Figure 5A. For the blank control group, very little bone formation was
found in the defect, whereas newly formed bone tissue was evident in
defects treated with COL or DP in a centripetal manner with regenerated
bone stretching from the margins toward the center. Quantitative
analysis of the micro-CT findings is presented in Figure 5B\&C. By week
8, the BMD of newly formed tissues in the control group remained as low
as 282.2\selectlanguage{ngerman}±16.17 mg HA ccm\selectlanguage{english}\textsuperscript{-1}. In contrast, the BMD
measurements were 529.3+-22.67 mg HA ccm\selectlanguage{english}\textsuperscript{-1} and
508.1+-14.96 mg HA ccm\textsuperscript{--1} for the COL and DP groups,
respectively, and the differences compared with the control group were
statistically significant difference (P=0.0266, n=3). For the ratio of
bone volume to total volume (BV/TV), similar trends were observed. Bone
regeneration was further evaluated histologically with Masson trichrome
staining (Figure 5D). In the control group, the defect areas showed poor
repair with mainly blue-stained fibrous connective tissues present. In
contrast, in the DP group, the defect was filled with woven bone (blue
stained) and cortical-like lamella bone (red stained), and a more mature
arrangement of collagen also was observed. Quantitative analysis
indicated the new bone percentage in the DP group (49.1\%+-7.69\%) was
significantly higher than that in the control group (12.2\%+-3.94\%;
P\textless{}0.01) and that in the COL group (31.5\%+-3.45\%;
P\textless{}0.05; Figure 5E). These results indicate that DP promoted
significantly more bone regeneration within the rat cranial defect GBR
model.

To evaluate the osteoimmunomodulatory properties of DP in the early
period of GBR, immunohistochemical staining for CD68 (pan marker of in
situ macrophages), iNOS (M1 marker), and CD206 (M2 marker) was performed
(Figure 5F). Semi-quantitative analysis of the staining results
indicated that a greater percentage of CD206+ M2 macrophages
(64.4\%+-7.64\%; P\textless{}0.05) was found in the DP group at week 1
compared with the other groups, whereas the number of iNOS+ M1
macrophages in the DP group was relatively low (33.6\%+-2.52\%;
P\textless{}0.05; Figure 5G). In summary, these results indicate that DP
induced polarization of more M2 phenotype macrophages in the early GBR
process.

\textbf{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.

\textbf{5 Conclusion}

In this study, DP derived from mini-pig cranium was successfully
fabricated. The present study then focused on the understanding of the
modulatory effects of DP in GBR by investigating the involvement of
macrophages in this process. The results indicate that DP scaffolds can
modulate macrophage polarization to a pro-healing M2 phenotype.
Additionally, macrophages polarized by the DP scaffolds supported BMSC
migration and differentiation in vitro. Even more importantly, DP
adequately improved the proportion of pro-healing macrophages as well as
the area of bone regeneration in a rat cranium critical defect GBR
model. Overall, our findings indicate that the DP can play an active
role in GBR via its immunomodulatory effects rather than its function as
a traditional barrier.

\textbf{Data Availability Statement:}

The data are available from the corresponding author on reasonable
request\textbf{.}

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\textbf{Table 1.} Primer sequences for qRT-PCR\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
Gene name & Forward (5' -- 3') & Reverse (5' -- 3')\tabularnewline
\midrule
\endhead
\emph{Gapdh} & ATGATTCTACCCACGGCAAG &
CTGGAAGATGGTGATGGGTT\tabularnewline
\emph{Arg1} & CGCCTCAAATCCAGCTGTAAG & GGGCCACAATCCAGTCGTT\tabularnewline
\emph{iNos} & TGGTGAAAGTGGTGTTCT & TTCCCTGTCTCAGTAGCA\tabularnewline
\emph{Runx2} & CCGAACTGGTCCGCACCGAC &
CTTGAAGGCCACGGGCAGGG\tabularnewline
\emph{Bmp2} & TCAAGCCAAACACAAACAGC & CCACGATCCAGTCATTCCA\tabularnewline
\bottomrule
\end{longtable}



\textbf{Figure Legends}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{FIGURE 1 ?} Confirmation of cell removal from decellularized
periosteum (DP).

\textbf{(A)} Macroscopic images of natural periosteum (NP) and
DP.\textbf{(B)} H\&E staining of NP and DP. Scale bar, 500 \selectlanguage{greek}μ\selectlanguage{english}m and 100
\selectlanguage{greek}μ\selectlanguage{english}m.\textbf{(C)} DAPI staining of NP and DP. Scale bar, 100 \selectlanguage{greek}μ\selectlanguage{english}m.
\textbf{(D)}DNA content quantification. \textbf{(E)} Collagen content
quantification. \textbf{(F)} GAG content quantification.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{FIGURE 2 ?} Characterization of DP.

\textbf{(A)} Representative SEM images and \textbf{(B)} AFM images
showing the surface topographies of collagen, NP, and DP
scaffolds.\textbf{(C)} Masson trichrome staining of each scaffold
type.\textbf{(D)} Picrosirius red analysis of the collagen fiber
structure in each group. \textbf{(E)} Immunohistochemical staining for
type I collagen. \textbf{(F)} Safranin-O staining for GAG
retention.\textbf{(G)} Water contact angle and waterdrop shape for each
scaffold type. \textbf{(H)} Mechanical properties of collagen, NP, and
DP scaffolds. (A,C,F) Scale bar, 200 \selectlanguage{greek}μ\selectlanguage{english}m and 50 \selectlanguage{greek}μ\selectlanguage{english}m. (D,E) Scale bar, 200
\selectlanguage{greek}μ\selectlanguage{english}m.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{FIGURE 3 ?} DP scaffolds regulate macrophage polarization in
vitro.

\textbf{(A)} Viability of BMSCs seeded on each scaffold type at the
indicated time points. \textbf{(B)} Viability of RAW264.7 cells seeded
on each scaffold type at the indicated time points. \textbf{(C)} Flow
cytometric analysis of macrophage population expressing CD86 (M1 type)
and CD206 (M2 type) at 3 days after seeding. \textbf{(D)} mRNA levels of
Arg-1 and iNOS in macrophages on the 3\textsuperscript{rd} day in
culture determined by qRT-PCR. \textbf{(E)} IL-10 and TNF-\selectlanguage{greek}α \selectlanguage{english}levels in
the supernatants on the 3\textsuperscript{rd} day in culture as detected
by ELISA. \textbf{(F)} Heatmap of the transcriptomes of RAW264.7 cells
after 3 days in culture on COL and DP scaffolds. \textbf{(G)} Volcano
plots of differentially expressed genes (DEGs) between the COL and DP
groups. \textbf{(H)} Gene Ontology (GO) enrichment analysis.\textbf{(I)}
Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{FIGURE 4 ?} Macrophages polarized in response to DP scaffold
promote BMSC migration and differentiation in vitro.

The viability of BMSCs after culture in different types of conditioned
medium (CM) for 1 and 3 days. \textbf{(B\&C)} Representative images and
quantitative analysis of BMSC migration after culture in different CM
for 1 day. Scale bar, 50 \selectlanguage{greek}μ\selectlanguage{english}m. \textbf{(D\&E)} Transwell-based BMSC
migration assays and quantification analysis. Scale bar, 1
mm.\textbf{(F)} ARS staining on the 21\textsuperscript{th} day in
culture. \textbf{(G)} Quantitative results of ARS staining on the
21\textsuperscript{th} day culture. \textbf{(H)} mRNA levels
of\emph{Runx2} and \emph{BMP2} in BMSCs after culture in different CM as
quantified by qRT-PCR.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{FIGURE 5 ?} In vivo bone regeneration with a rat critical-sized
defect as a guided bone regeneration (GBR) model.

\textbf{(A)} Three-dimensional construction and representative micro-CT
images for each scaffold group. Scale bars, 1 mm and 5mm. \textbf{(B and
C)} Quantitative results for BMD and BV/TV. \textbf{(D)} Representative
images of Masson trichrome staining of samples collected in the 8th week
post-surgery. Scale bar, 200 \selectlanguage{greek}μ\selectlanguage{english}m. \textbf{(E)} Quantitative results for
percentage of new bone growth. \textbf{(F)} Immunohistochemical staining
for macrophage polarization pan marker CD68, M1 marker iNOS, and M2
marker CD206 in tissue filling the defect tissues. Positively stained
cells are indicated by arrows. Scale bar, 100 \selectlanguage{greek}μ\selectlanguage{english}m.
\textbf{(G)}Quantification of positively stained cells.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Scheme 1 ?} Schematic illustration of bone regeneration in the
GBR model promoted by DP via regulation of macrophage polarization
towards the M2 phenotype.

\selectlanguage{english}
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