Preparation and Evaluation of Tibia- and Calvarium-Derived

Article pubs.acs.org/journal/abseba Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX-XXX

Preparation and Evaluation of Tibia- and Calvarium-Derived Decellularized Periosteum Scaffolds Jianfeng Zhang,† Qi Zhang,† Jiaxin Chen,† Jinhu Ni, Zeng Zhang, Gangliang Wang, Liyang Song, Shunwu Fan,* Pengfei Chen,* and Xianfeng Lin* Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Medical College of Zhejiang University, 3 East Qing Chun Road, Hangzhou, Zhejiang Province 310016, P.R. China S Supporting Information *

ABSTRACT: The periosteum plays a key role in bone regeneration and an artificial bionic material is urgently required. The periostea on the tibia and skull differ with respect to the types of cells, microstructure, and components, leading to different biological functions and biomechanical properties. We aimed to prepare decellularized periosteum scaffolds derived from different origins and evaluate their angiogenic and osteogenic activities. Histological assessment of α-smooth muscle actin, bone morphogenetic protein-2, and alkaline phosphatase in tibial and calvarial periosteum tissues provided preliminary information on their differing angiogenic and osteogenic properties. We developed decellularization protocols to completely remove the periosteum cellular components and for good maintenance of the hierarchical multilayer structures and components of the extracellular matrix (ECM) with no cytotoxicity. Moreover, using a chicken egg chorioallantoic membrane assay and a nude mouse implantation model, we found that tibia-derived periosteum ECM had superior osteogenic activity and calvarium-derived ECM had good angiogenic activity. The preliminary mechanisms of differing activities were then evaluated by osteogenesis- and angiogenesis-related gene expression in human umbilical vein endothelial cell- and MC-3T3 cellseeded ECM scaffolds. Thus, this study provides periosteum biomaterials that are derived from specific tissues and have different functional properties and structures, for use in bone regeneration.

KEYWORDS: decellularized, tibial periosteum, calvarial periosteum, angiogenesis, osteogenesis, bone regeneration

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formed by intramembranous ossification.4 The periosteum on the tibia possesses good mechanical properties5 and has remarkable osteogenic activity4,6 due to superior thickness and higher numbers of osteoblasts. However, the osteogenesis

INTRODUCTION The periosteum, a dense connective tissue that adheres to bone surfaces, has satisfactory osteogenic activity.1 Animal experiments and clinical studies have demonstrated that the periosteum plays a key role in bone regeneration. 2,3 Furthermore, a recent study showed some differences between tibial and calvarial periostea. In the embryonic stage, limbs are formed by endochondral ossification, whereas calvaria are © XXXX American Chemical Society

Received: August 2, 2017 Accepted: October 15, 2017 Published: October 16, 2017 A

DOI: 10.1021/acsbiomaterials.7b00548 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

preliminary investigation of the osteogenesis and angiogenesis mechanisms by seeding human umbilical vein endothelial cells (HUVECs) and MC-3T3 cells onto tibial and calvarial decellularized scaffolds.

in the limbs (tibial) periosteum is instable and the surface of the new bone is rough, whereas the surface of the new bone formed by the calvarial periosteum is relatively smooth,6 which is mainly attributed to the corresponding original structures and functions. Bone defects caused by trauma, necrosis, or deformity are common orthopedic conditions and are major concerns for orthopedic surgeons. The disability induced by orthopedic injuries has increased by 34% from 1990 to 2010 and accounts for heavy economic burdens.7 To obtain the superior osteogenic activity performed by the periosteum, native-derived or synthetic periosteum biomaterials may be a promising option for enhancing bone regeneration. A trend in recent years is to imitate the three-dimensional structure of the native periosteum to produce artificial periosteum materials.8 Polyethylene glycol-based hydrogels seeded with mesenchymal stem cells (MSCs) and osteoprogenitor cells in a 50:50 ratio were used as the periosteum to successfully improve murine segmental femoral graft healing in vivo.9 Zhao et al. used tissue-engineered periosteum fabricated with osteo-induced rabbit bone marrow MSCs and porcine small intestinal submucosa for allograft implantation to treat segmental bone defects.10 Another study demonstrated that a combination of (a) polydimethylsiloxane substrates with a nanopattern coating of bovine collagen I and (b) the osteogenic potential of human MSCs in vitro would be beneficial for engineering an artificial periosteum.11 However, these engineered periostea cannot completely imitate the microstructures and functions of the periosteum; they also have associated issues such as long preparation time, limited vascularization, and limited cell-penetration ability. Moreover, these issues increase the degree of difficulty experienced in imitating the specific periosteum because the structures and functions differ between the different types of periostea, such as the tibial periosteum and calvarial periosteum. Therefore, it is necessary to fabricate specific periostea with well-maintained native structures and components even to meet different requirements in the clinical setting. Decellularization, a novel type of biomaterial preparation technology, may solve the aforementioned hurdles. This technique can be used to remove cells from specific tissues and to retain the complex native extracellular matrix (ECM) consisting of structural and functional biological components.12,13 Recently, the efficiency and safety of decellularized scaffolds have been demonstrated in various tissues, such as skeletal muscles,14 the cartilage,15 and the dermis.16 Some kinds of scaffolds have been widely used for tissue engineering or even in the clinic.17 In our previous studies, we prepared a tibial periosteum ECM scaffold and evaluated its biological and structural properties; we found that the acellular tibial periosteum can be considered as a biomimetic scaffold that permits adherence, migration, and proliferation of pluripotent cells in vitro.18 Therefore, this study aimed to analyze differences in the functional properties and the structures of tibial and calvarial periostea. This analysis involved the following: (a) preparation of tibial and calvarial decellularized periostea and evaluation of their biological, structural, and biomechanical properties; (b) assessment of the cytotoxicity of the periosteum ECM scaffold; (c) evaluation of the angiogenic activity of the bioscaffolds by using a chicken egg chorioallantoic membrane (CAM) assay in vitro; (d) assessment of osteogenic activity by implantation of MSC-seeded decellularized periostea in nude mice; and (e)

2. MATERIALS AND METHODS 2.1. Overview of Study Design. Native tibial and calvarial periostea were harvested and decellularized using physical, chemical, and enzymatic methods. The corresponding decellularized tibial and calvarial periostea were evaluated for cellular component removal, major biological components, biomechanics, 3D structure, and angiogenic and osteogenic activities. All animal materials were obtained from the Animal Experimental Centre of Zhejiang University. All animal experiments were approved by the Animal Experimental Ethics Committee of Sir Run Run Shaw Affiliated Hospital, Zhejiang University School of Medicine, and the animals were treated according to the approved experimental protocols. 2.2. Harvesting of Native Tibial and Calvarial Periostea. A total of 56 male New Zealand white rabbits (age, ∼4 months; weight, 2−2.5 kg) were anaesthetized using sodium pentobarbital (30 mg/kg). Under sterile conditions, the skin and overlying fascia at the anteromedial site of both proximal tibiae were opened to expose the tibial periosteum, those at the forehead and calvarium were opened to expose the calvarial periosteum and those at the forehead and calvarium were opened to expose the calvarial periosteum. Sections (diameter, 6 mm) of the tibial and calvarial periostea were removed using sharp subperiosteal dissection and then immediately rinsed with phosphate-buffered saline (PBS; Hyclone, USA) twice to remove the blood on the surface. The periostea were stored in PBS containing 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, USA) at 4 °C until use (usually within 7 days). 2.3. Decellularization Process. All decellularization treatments were performed at 25 °C under agitation (100 rpm) with an orbital shaker (Qilinbeier, China). Only the native calvarial periostea (NCPs) were immersed in 30% acetone (v/v) for 4 h and then rinsed with PBS for 2 h before freeze−thaw cycling. First, the samples were frozen using liquid nitrogen and thawed at 25 °C for three cycles. Then, the two kinds of samples were separately treated with Triton-X100 (1% v/ v) for 12 h and with 10 g/L sodium dodecyl sulfate (SDS; Sigma, USA) solution for 2 h to further remove the cellular and nuclear components, and then rinsed with PBS for 2 h. To further minimize the DNA content, the samples were treated with a mixed solution containing 0.05 U/L DNase I and 0.001 U/L RNase A (TaKaRa, Japan) at 37.5 °C for 12 h and then rinsed under running ultrapure water for 1 day. Finally, the samples were placed in PBS at 4 °C for preservation. Acetone can be cleared after sufficient rinsing, and is widely used to remove cells and cellular components during decellularization.19 The decellularization process flowchart is shown in Figure S1. 2.4. Evaluation of the Potential Angiogenic and Osteogenic Properties of the Native Periosteum. Immunohistochemical staining of vessel and osteogenesis-related proteins, that is, α-smooth muscle actin (α-SMA), vascular endothelial growth factor (VEGF), bone morphogenetic protein-2 (BMP-2), alkaline phosphatase (ALP), and collagen X was performed. Briefly, sections were rehydrated and incubated overnight at 4 °C with the following antibodies against these proteins: α-SMA (1:200, PL Laboratories, Canada), BMP2 (1:200, Abbiotec, USA), VEGF (1:800), collagen X (1:200, YANMU, China), ALP (1:50, LSBio, China). This was followed by incubation with secondary antibodies for 2 h at room temperature. After the sections were rinsed thrice in PBS, they were incubated with diaminobenzidine for 10 min at room temperature. The nuclei were visualized by staining with hematoxylin or 4′,6-diamidino-2-phenylindole (DAPI; Sigma, USA). The sections were examined by light and fluorescence microscopy. α-SMA staining was performed for detection of smooth muscle cells in periosteal blood vessels, and VEGF staining, for analysis of the potential angiogenic activity of tibial and calvarial periosteal.20 BMP-2 is one of the most important regulators for promoting osteogenesis and acts on progenitor cells to induce differentiation into B


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ACS Biomaterials Science & Engineering osteoblasts, whereas ALP is a marker for osteoblast expression and osteogenic activity.21 Collagen X is a scaffold protein and is closely related to osteogenesis.22 2.5. Evaluation of Cellular and Nuclear Removal for Decellularized Periosteum ECM. 2.5.1. Histological Analysis. The native tibial periosteum (NTP), NCP, decellularized tibial periosteum (DTP), and decellularized calvarial periosteum (DCP) samples were fixed in 10% formalin, embedded in paraffin, and sectioned into 5 mm-thick slices. The sections were deparaffinized, rehydrated, and washed in distilled water. Histological analysis using hematoxylin and eosin (H&E) staining was performed to observe cellular components and the general structure of the ECM in all of the above sample groups. 2.5.2. Qualitative and Quantitative DNA Analyses. Slides prepared from the paraffin-embedded samples were used for DAPI staining to detect the presence of any residual nuclear components. For further assessment of nucleic acid concentration, lysates of samples from all four groups (n = 8 in each group) were prepared by digestion with a proteinase K and RNase A (TaKaRa) mixed solution at 56 °C for 6 h in a water bath until there were no visible materials; then, we extracted nucleic acids according to the Takara DNA kit instructions (TaKaRa). The DNA concentrations were measured using a spectrophotometer (Bio-Rad, USA), and the measured DNA quantity was normalized to the initial dry weight of the tissues. Then, the remaining purified nucleic acid sample was collected with an elution buffer. The size of the extracted DNA fragments was determined using 2% agarose gel electrophoresis. 2.6. Evaluation of the Remaining Biological Components of Decellularized Periosteum ECM. 2.6.1. Qualitative and Quantitative Glycosaminoglycan (GAG) Analyses. Safranin O staining was performed for qualitative GAG analysis. Samples (n = 10 per group) were washed twice with PBS, freeze-dried, and sheared into pieces. As per the manufacturer instructions for the Blyscan Sulfated Glycosaminoglycan Assay kit (Biocolor, UK), samples were added to 300 μL papain solution (containing 10 mmol/L L-cysteine-HCl, 10 mmol/L EDTA-2Na, 5 mg/mL papain); subsequently, the pH was adjusted to 6.5 and the samples were treated in a water bath at 65 °C for 12 h for digestion. After 5 min, the optical density of a mixture of 250 μL of the treated sample and 2.5 mL 1, 9-dimethyl phosphite methylene blue (DMMB) solution (containing 16 mg/L DMMB, 3.04 g/L glycine, 2.37 g/L NaCl, pH 2) was measured at 525 nm. The standard concentration used for chondroitin sulfate was 100 μg/mL; the standard was diluted to 80, 60, 40, and 20 μg/mL to obtain a standard curve. GAG content was estimated against a chondroitin sulfate standard curve ranging from 100 μg/mL to 0 μg/mL. 2.6.2. Qualitative and Quantitative Collagen Analyses. Masson trichrome staining and immunofluorescent staining of collagen X were performed to observe specific collagen distribution and orientation. The collagen content was determined on the basis of the hydroxyproline (Hyp) content, which provides an accurate and reliable estimate of the collagen content of tissues;23 Hyp content was quantified using the Hyp assay kit (Keygen, China) according to the manufacturer’s instructions. First, samples from the four groups (n = 15 in each group) were lyophilized to a constant weight, followed by acid hydrolysis with 0.1 mol/L hydrochloric acid at 100 °C for 20 min and neutralization with sodium hydroxide. Then, the absorbance was measured at 570 nm with an Epoch Microplate Spectrophotometer (BioTek, USA). The Hyp levels were determined on the basis of standard samples from the assay kit. The total collagen content per milligram dry weight of the samples was calculated using a Hyp-tocollagen ratio of 1:7.46. 2.7. Biomechanics Evaluation of Decellularized Periosteum ECM. The biomechanical properties of the NTP, NCP, DTP, and DCP (n = 5) were evaluated with uniaxial tensile tests performed using an SMT1−50N Force Transducer (Interface, USA). Samples (length, 10 mm) were immersed in PBS until testing began. They were clamped to the grips in the mechanical apparatus, and the initial specimens’ width and thickness were recorded. The samples were then stretched to tensile failure at a rate of 10 mm/min. The stress−strain curves were obtained. The ultimate stress (UTS) was calculated by

dividing the maximum load by the cross-sectional area of the specimen. The strain at failure was calculated by dividing the change in length by the initial length of the specimens. The elastic modulus was calculated from the slope of the ascending linear region of the stress−strain curve. 2.8. Evaluation of the Periosteum Microstructure. To evaluate the matrix structure after decellularization, samples from four groups (n = 4 in each group) were fixed using 3% glutaraldehyde in PBS for 1 h, followed by 0.1 M sodium cacodylate buffer (applied three times for 5 min each). After the specimens were rinsed in cacodylate buffer, they were dehydrated through an ethanol gradient (30, 50, 70, 80, 95, and 100% ethanol for 10 min each) and were dried in hexamethyldisilazane (HMDS; Sigma, USA). Subsequently, these dry samples were coated under vacuum with a platinum alloy at a thickness of 25 nm and were immediately flash carbon coated under vacuum. The samples were observed with an S-3000N scanning electron microscope (Hitachi, Japan). Oil Red O staining was performed for analysis of adipose cells. The frozen samples were sectioned into 5-μm sections and air-dried for 5 min; subsequently, the slides were soaked in absolute propylene glycol for 3 min and then stained with Oil Red O solution. The images were obtained for light microscope (NIKON Eclipse ci, Japan). 2.9. MSC Isolation and Culture. All procedures were conducted under institutional review board approval and in accordance with the research guidelines of Zhejiang University. The harvested bone marrow samples from the ilia were collected from healthy male New Zealand white rabbits (n = 4); lymphocyte separation medium (Sangon Biotech, China) was carefully added and the mixture was centrifuged at 3000 rpm for 30 min at 37 °C. After centrifugation, the mixture separated into three layers; the middle transparent oily layer contained the MSCs. The middle-layer liquid was transferred into the cell culture dish and cultured with Dulbecco’s modified Eagle’s medium/HAMF12 (DMEM/F12, Gibco) containing 20% fetal bovine serum (FBS; Gibco), 100 U/ml penicillin, and 100 μg/mL streptomycin (Gibco) at 37 °C in a humidified incubator (Thermo, USA) with 5% CO2. 2.10. Evaluation of Cytotoxicity of Decellularized Periosteum ECM Scaffolds. 2.10.1. Preparation of ECM Scaffold Leaching Solution. Standard-sized (2 × 2 mm) DCP and DTP samples were immersed in 0.1% peracetic acid for disinfection and rinsed with PBS more than five times; the surface water was removed using clean sterile gauze. The leaching solution was generated using 100 mg dry weight of samples (decellularized calvarial and tibial periosteum ECM scaffold) incubated in 1 mL of DMEM-HG medium at 37 °C for 48 h; the supernatant was collected as leaching solution (100%). The control group was the standard DMEM-HG medium. 2.10.2. Assessment of ECM Scaffold Cytotoxicity. The cells were seeded in 96-well cell culture plates with DMEM/F12, at a concentration of 5 × 103 cells/well. After 24 h of culture, the medium was removed and replaced with different concentrations (25, 50, and 100%) of leaching solutions. Cells cultured in only the standard medium served as the control group. The metabolic activity of the cells was measured using Cell Counting Kit-8 (CCK-8, Dojindo, Japan), and the data were recorded on days 1, 3, 5, and 7. The absorbance was measured at 450 nm with an Epoch Microplate Spectrophotometer. Five replicates were used for each sample. Cell viability after treatment with standard DMEM/F12 and 100% leaching solution was evaluated using the Live−Dead Cell Staining Kit (BioVision, USA) on days 1 and 3 according to the manufacturer’s instructions. Briefly, the adherent cells were washed with PBS and were then exposed to staining solution (mixing 1 μL of 2.5 mg/mL propidium iodide and 1 μL of 1 mM Calcein-AM in 1 mL of Staining Buffer) for 15 min at 37 °C. The cells were observed immediately using a Nikon ECLIPSE 80i microscope (Nikon, Japan); the living cells showed green fluorescence. 2.11. Chicken Egg CAM Model. The angiogenic capacity of scaffolds was quantified by the chicken egg CAM assay as previously described.24 We used the chicken egg CAM model to evaluate the angiogenic activity of the DTP and DCP ECM scaffolds. Fertilized chicken eggs were incubated at 37 °C for 7 days; then, an oval window (diameter, 1.5 cm) was cut into the shell with small dissecting scissors to expose the embryo and CAM vessels. Then, 4 × 4 mm2 native and C


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Figure 1. Immunohistochemical staining of natural tibial and calvarial periostea to analyze differences in their angiogenic and osteogenic properties. α-SMA, BMP-2, and ALP staining of (A, C, E) natural tibial and (B, D, F) calvarial periostea. Scale bar, 100 μm. Red arrow, blood vessel; green arrow, ALP-positive staining.

Figure 2. Complete removal of the cellular components of tibial and calvarial periostea. Macroscopic images of the (A1) NTP, (A2) NCP, (A3) DTP, and (A4) DCP. (B1−4) H&E staining, (C1−4) DAPI staining, (D) agarose gel electrophoresis, and (E) DNA quantification for the above four groups. Zone I and zone II stand for the cambium and fibrous layers, of tibial periosteum; zones III, IV, and V stand for cambium, adipose, and fibrous layers, respectively, of the calvarial periosteum. Scale bar, 100 μm. *** p < 0.001. 2.12. Subcutaneous Implantation of MSC-Seeded ECM Scaffolds. MSCs were seeded into DTP and DCP scaffolds (n = 12 for each group) at a concentration of 5 × 106 cells/scaffold and incubated for 1 week. Twelve adult nude mice (age, 3 weeks) were equally divided into two groups. Under general anesthesia and sterile conditions, 5 mm incisions were made and MSC-seeded DTP and DCP scaffolds were implanted into either side of the dorsal midline subdermal pockets. The incisions were then closed and each mouse

decellularized periostea and gelatin sponges soaked in PBS (negative control) or 400 ng/mL VEGF (positive control) were placed on the CAM among the blood vessel branches. The samples were examined daily until 7 days, following which they were photographed and evaluated using a stereomicroscope to count the blood vessels surrounding the implants. The blood vessels that were less than 100 μm in diameter and converged toward the tissues placed were counted blindly by three assessors (n = 4 for each group). D


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Figure 3. Preservation of the biological components of the tibial and calvarial periosteum ECM scaffolds. (A1−A4) Masson trichrome and (B1−B4) Safranin O staining of tibial and calvarial periostea before and after decellularization. (C1−C4) Collagen X immunofluorescence cell staining of NTP, DTP, NCP, and DCP. Quantitative testing of collagen and GAGs (D and E) in tibial and calvarial periostea before and after decellularization. Zone I and zone II stand for the fibrous and cambium layers of tibial periosteum, respectively; Zones III, IV, and V stand for fibrous, adipose, and cambium layers of the calvarial periosteum, respectively; yellow arrows stand for Collagen X. Scale bar, 100 μm. ns: p > 0.05; **: p = 0.001−0.01; ***: p < 0.001. was allowed unrestricted activity in the cage. During the first and second weeks, three mice from each group were sacrificed and the implants were harvested and processed for histologic analysis; 5-μmthick sections were prepared and subjected to HE staining, BMP-2 staining, and von Kossa staining. 2.13. Evaluation of the Osteogenesis and Angiogenesis Mechanisms for Tibial and Calvarial Decellularized Scaffolds. 2.13.1. Seeding HUVECs and MC-3T3 Cells into Scaffolds. HUVECs and MC-3T3 cells (osteoblast lineage) were separately obtained from Lonza Inc. and ATCC, and cultured under routine conditions. These cells were seeded into DTP and DCP scaffolds (n = 5 for each group) at a concentration of 5 × 106 cells/scaffold and incubated for 3, 7, and 14 days. Cells from the same source were seeded in a Petri dish at a concentration of 5 × 105 cell/mL, as the control group. 2.13.2. Osteogenesis- and Angiogenesis-Related Gene Expression of Cell-Seeded Scaffolds. Real-time polymerase chain reaction (RTPCR) was performed on RNA extracted from cell-seeded (both HUVECs and MC-3T3 cells) DTP and DCP scaffolds to evaluate the gene expression levels of collagen I, BMP-2, Runt-related transcription factor (Runx2), osterix (OSX), VEGF, hypoxia-inducible factor 1alpha (HIF1-α), and angiopoietin-1 (Ang-I). The forward and reverse primers are shown in Table S1 (detailed see the Supporting Information). RNA from scaffolds and monolayer controls was isolated using the RNeasy Mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. RNA integrity and quantification were tested with NanoDrop 8000. The RNA was then reverse transcribed, and after amplifying the cDNA, RT-PCR was

performed on a 96-well plate ABI Prism 7500 system (Applied Biosystems, Foster City, CA, USA) with the SsoFast EvaGreen supermix (Bio-Rad, Hercules, CA, USA). The expression ratio for each gene was quantified mathematically by the 2−ΔΔCt method, using GAPDH as the housekeeping gene for mouse cells and β-actin for human cells, and the results for the target genes were compared with those for the control groups. 2.14. Statistical Analysis. Quantitative results have been represented as mean ± standard deviation values. One-way analysis of variance (ANOVA) was performed to analyze the differences in the DNA content, Hyp content, OD values, number of vessels, the mechanical properties of the periostea and gene expression levels. Statistical analysis was performed using the SPSS 16.0 software (SPSS Inc., Chicago, USA), and significance was defined as p < 0.05.

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RESULTS

3.1. Histological Assessment of the Angiogenic and Osteogenic Activities of the Native Periosteum. α-SMA staining of the native periosteum revealed that the NCP had a higher vessel density than the NTP and that the vessels were mainly located in the fibrous layer (Figure 1A, B). Interestingly, BMP-2 staining showed that the NTP contained more BMP-2 than the NCP, indicating that the periosteum derived from the tibia had stronger osteogenesis-promoting activity (Figure 1C, D). We also found that the BMP-2 was concentrated in the E


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ACS Biomaterials Science & Engineering superficial cambium layer and the entire fibrous layer. Moreover, the cells that adhered in the cambium of the NTP had a higher ALP content (Figure 1E, F). 3.2. Complete Removal of the Cellular and Nuclear Components of the Decellularized Periosteum ECM. The macroscopic images obtained before and after decellularization are shown in Figure 2A1 and A4. The color of the NTP and NCP samples changed from red to milky white after the decellularization process. H&E staining showed the apparent layers and difference in the cellularity in the NTP and NCP: The NTP could be divided into two layers, that is, the cambium (Figure 2B1 I) and fibrous layers (Figure 2B1 II), whereas NCP can be divided into three layers including cambium, adipose, and fibrous layers (Figure 2 B2 III, IV and V respectively). We found that the cellular nuclei were deeply stained and polymorphic in the cambium of the NTP. In the fibrous layer, fibrocytes were distributed among the dense and thick fiber bundles. In the NCP, the density and number of cambium cells were lesser and the nuclei were stained lighter than those in the NTP, suggesting that the osteogenic activity was weak. The middle layer was filled with a large number of adipose cells, among which several blood vessels were crossed; unlike the finding for the NTP, the fiber bundle in the fibrous layer of the NCP was relatively slight and loose. H&E staining also demonstrated that the cells were mainly removed and that the ECM scaffold was well preserved in the DTP and DCP scaffolds (Figure 2B1−B4). DAPI staining also proved that hardly no nuclear components remained after decellularization (Figure 2C1−C4). Furthermore, there were no detectable bands of DNA fragments after separation on a 2% agarose gel, compared with the native periostea (Figure 2D). The DNA quantification results suggested that more than 95% of the nuclear material was eliminated by the decellularization processes at both the tibial (25.40 vs 1315.12 ng/mg, p < 0.001) and calvarial (8.86 vs 265.33 ng/mg, p < 0.001) periosteum ECM scaffolds. The DNA content in the NCP was significantly lesser than that in the NTP (p < 0.001; Figure 2E). 3.3. Good Preservation of the Major Biological Components of the Periosteum ECM after Decellularization. The Masson trichrome staining results indicated that the periosteal ECM was primarily composed of collagen bundles and fibers. The tibial periosteum was much thicker and denser than the calvarial periosteum. The integrity and continuity of DTP collagen fibers were well maintained, but the DCP underwent slight changes and became more loose and porous (Figure 3A1−A4). Immunofluorescence staining showed that the cells adhered in the cambium layer of the NTP showed high collagen X expression levels, whereas those in the NCP showed low expression levels (Figure 3C1, C3). After decellularization treatment, collagen X has a decline in some degree (Figure 3C2, C4). In both tibial and calvarial periostea, the collagen X in the fibrous layer was absent; it was evenly distributed in the adipose layer of the NCP. After the decellularization process, the collagen X content seemed to decrease to some extent. Further quantitative analysis of collagen was performed by determining the hydroxyproline content (Figure 3D). The collagen content of both the DTP (57.09 vs 65.78 μg/mg, p > 0.05) and DCP (13.83 vs 16.55 μg/ mg, p > 0.05) did not significantly decrease on decellularization. Safranin O staining showed that the GAGs were well identifiable both before and after decellularization (Figure 3B1−B4). On decellularization, the staining became a little light in both tibial and calvarial periosteum ECM scaffolds, which

indicated that the GAG content relatively decreased. GAG quantification showed that the amounts of GAGs in tibial (2391.04 vs 4013.79 ng/mg, p < 0.001) and calvarial (1260.23 vs 1747.53 ng/mg, p < 0.01) periosteum ECM scaffolds decreased to some extent (Figure 3E) but were mainly preserved. In the tibial periosteum groups, the collagen and GAG content was greater than that in the calvarial groups. 3.4. Differing Structures of the Tibial and Calvarial Periostea. Oil Red O staining showed that the NCP was mainly composed of adipose cells in the middle layer (Figure 4B), whereas these were absent in the NTP (Figure 4A). Views

Figure 4. Preservation of the hierarchical multilayer structures of tibial and calvarial periosteum ECM scaffolds. Oil Red O staining of natural (A) tibial and (B) calvarial periostea. SEM images of (C, D, G, H, K, L) tibial and (E, F, I, J, M, N) calvarial periostea before and after decellularization. The arrow indicates an adipocyte. (A−F) Scale bar, 100 μm; (G, H, K−N) Scale bar, 30 μm; I-J: scale bar, 5 μm.

of the entire aspects of the NTP, DTP, NCP, and DCP, obtained using SEM analysis, are shown in Figure 4C−F. Dense massive bundles of collagen were noted in the fibrous layer of the tibial periosteum (Figure 4K); after decellularization, the collagen bundles became slightly loose (Figure 4L). In the calvarial periosteum, adipose cells were present in the middle layer (Figure 4M) but were absent in the collagen skeleton after decellularization (Figure 4N). Interestingly, on the surface of the cambium layer, large bundles of the collagen from the tibial periosteum were present; however, compared with the calvarial periosteum, the surface of the cambium layer was smoother and the collagen bundles were more slender (Figure 4G, I). After decellularization, cells were removed and the loose and porous collagen scaffold remained in the cambium layers of both tibial and calvarial periostea (Figure 4H, J). 3.5. Differing Biomechanical Properties of the Tibial and Calvarial Periostea. Representative uniaxial tensile F


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Figure 5. (A−C) Elastic modulus, ultimate stress, and strain failure of NTP, DTP, NCP, and DCP. ns: p > 0.05, *: p < 0.05.

Figure 6. Cytotoxicity evaluation of decellularized periosteum scaffolds. The metabolic activity of cells cultured in the standard medium (control group) and leaching solutions used at different concentrations (25%, 50%, and 100%) in the (A) tibial and (B) calvarial periosteum ECM scaffolds on days 1, 3, 5, and 7. (C) Live−Dead cell staining of 100% leaching solution of tibial and calvarial periosteum ECM scaffolds on days 1 and 3. Scale bar, 25 μm.

3.6. Cytotoxicity of Decellularized Periosteum Scaffolds Was Not Confirmed. The CCK-8 assay and Live−Dead cell staining were performed to confirm that there was no chemistry and no enzyme residues in the decellularized scaffolds. The CCK-8 test indicated that the leaching solutions, used at different concentrations, had no obvious impact on cellular metabolic activity and that the OD values gradually increased during the culture periods (p > 0.05; Figure 6A, B). Live−Dead cell staining also showed favorable proliferation trends of MSCs in all groups (Figure 6C). 3.7. Differences between the Angiogenic and Osteogenic Effects of the Tibial and Calvarial Periosteum ECM. Immunohistochemical staining revealed that the native periosteum contained large amounts of VEGF; in both tibial and calvarial periostea (Figure 7A1, A3). After the decellularization process, VEGF significantly decreased and was hardly

stress−strain curves for the NTP, DTP, NCP, and DCP are shown in Figure S2. The elastic modulus of decellularized tibial (24.81 ± 1.48 vs 24.99 ± 1.32 MPa, p = 0.1789) and calvarial (4.55 ± 0.70 vs 5.56 ± 0.75 MPa, p = 0.057) periostea ECM scaffolds was not significantly affected compared to that of the native groups. However, the UTS of the calvarial periosteum (5.21 ± 0.60 vs 3.32 ± 0.47 MPa, p < 0.05) slightly decreased after decellularization and was absent in tibial groups (15.63 ± 0.93 vs 15.38 ± 0.93 MPa, p = 0.693). In addition, the strain failure of both tibial and calvarial periostea was comparable to that of the native periostea (1.66 ± 0.07 vs 1.68 ± 0.11 MPa, p = 0.441; 0.93 ± 0.05 vs 0.97 ± 0.10 MPa, p = 0.681, respectively). Notably, the biomechanical properties of the tibial periosteum were superior to those of the calvarial periosteum before and after decellularization (Figure 5). G


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Figure 7. Differing angiogenic properties of tibial and calvarial periostea before and after decellularization. VEGF staining was performed for the (A1) NTP, (A2) DTP, (A3) NCP, and (A4) DCP. Qualitative (B1−6) and quantitative (C) analyses of the above four groups (B3−6) with the CAM model, as compared to the positive (B1) and negative (B2) control groups. (A1−A4) scale bar, 100 μm; (B1−B6) scale bar, 5 mm. *: p = 0.01−0.05; **: p = 0.001−0.01; ***: p < 0.001.

observed in the DTP group (Figure 7A2, A4). By contrast, the DCP group had some sporadic positive staining. The CAM model was used to further analyze the angiogenic activity of the four periosteum groups, especially the ECM scaffolds; the detailed results are shown in Figure 7B1−B6. The macroscopic images showed that the periosteum (all four groups) induced a greater number of new blood vessels than that noted in the PBS group, but the number was lesser than that for the VEGF group. The calvarial periosteum induced more new blood vessels than the tibial periosteum, both before and after decellularization (Figure 7C). The above findings showed that the DCP scaffold may possess better angiogenesis activity than the DTP scaffold. The DTP and DCP reseeded with MSCs were embedded into the dorsal subcutaneous spaces of the mice for 2 weeks and all these mice survived. H&E staining revealed that, as time progressed, an increasing number of seeded cells proliferated and migrated into the DTP and DCP scaffolds (Figure 8A−D). Furthermore, BMP-2 and von Kossa staining was used to evaluate the effects of osteogenesis promotion. On day 7, the DTP and DCP showed negative staining for BMP-2 (Figure 8E, G); positive results were noted on day 14 in the MSC-seeded scaffolds (Figure 8F, H, M). The cells in the DTP group exhibited more prominent osteogenesis than those in the DCP group. Qualitative analyses involving von Kossa staining also illustrated that both MSC-seeded tibial and calvarial periosteum ECM scaffolds showed biomineralization and that a greater number of calcium nodes were formed in the cambium layer in the DTP group on day 14 and more vessel formation was noted in the DCP group on day 14 (Figure 8I−L, O, N). The RT-PCR results for DTP and DCP ECM scaffolds seeded with HUVECs and MC-3T3 cells are shown in Figure 9. We found that, compared to the control groups, the tibial- and calvarial-derived scaffolds were more beneficial for HUVEC (angiogenesis)- and MC-3T3 (osteogenesis) cell-related gene expression. The HUVECs-seeded DCP showed higher VEGF, HIF-1α, and ANG-1 expression than that seen in the DTP and

monolayer groups on day 14 (Figure 9A−C). BMP-2, OSX, and collagen I expression showed an evident increase in the MC-3T3 cell-seeded DTP at a later stage, and RUNX2 expression also showed trend for stable increase (Figure 9D− G). The interaction between these cytokines during the angiogenesis and osteogenesis has been shown in Figure S3.

4. DISCUSSION In this study, we comprehensively evaluated periostea derived from different origins, both before and after decellularization. Differentiation of the angiogenic and osteogenic activities of the DTP and DCP was noted in the CAM and nude mouse implantation models. Further, preliminary mechanisms underlying the effect of tibial and calvarial periosteum scaffolds in osteogenesis and angiogenesis were evaluated on the basis of the related gene expression in HUVECs and MC-3T3-E1 cells. We found that the NTP was more active in promoting bone regeneration, whereas the calvarial periosteum promoted angiogenesis. The tibial periosteum has two layers: outer layer (fibrous layer) and inner layer (cambium layer). The fibrous layer consists of numerous fibroblasts and collagen fibers with scarce blood vessels in the ECM.25 The cambium layer in the tibial periosteum contains several more cell layers than the calvarial periosteum, including osteoblasts and preosteoblastic cells.26 The increased cell density and polymorphisms found in the NTP in the current study confirm this. The calvarial periosteum consists of three layers: outer fibrous layer, middle adipose layer, and inner cambium layer. In the middle adipose layer, many adipose cells are found embedded in collagen fibers and could promote differentiation of epithelial cell and enhance vasculogenesis.27 In addition to these differences in cellular distribution (more cells in the cambium layer) and morphological features (more condensed and massive collagen fibers), the superior biomechanics (including elastic modulus, UTS, and strain failure) and higher collagen content (including collagen I and collagen X) of the tibial H


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Figure 8. Nude mouse implantation model of tibial and calvarial periosteum ECM scaffolds reseeded with MSCs. (A−D) H&E, (E−H) BMP-2, and (I−L) von Kossa staining of tibial and calvarial periosteum ECM scaffolds on days 7 and 14. Relative quantity of (M) BMP-2, (N) calcium accumulation and (O) vessel formation on day 7 and 14. The arrow indicates a blood vessel, *, intimal layering. (A−D) Scale bar, 100 μm; (E−L) Scale bar, 50 μm. ns: p > 0.05; *, p = 0.01−0.05; **, p = 0.001−0.01; ***, p < 0.001.

periosteum scaffold could also facilitate bone regeneration.22,28,29 In the case of biological components, BMP-2, ALP, and collagen X are closely related to osteogenic differentiation and biomimetic mineralization.30,31 Positive α-SMA staining indicates that the blood vessel components and high VEGF expression may be responsible for new bone tissue regeneration.32 The stronger osteogenic activity of NTP in our study is consistent with the findings of a previous study18 and may be attributed to increased BMP-2, ALP, and collagen X expression. And collagen X enriched in its cambium layer is benefit for cells osteogenic differentiation. The obviously angiogenic effect of NCP can also be attributed to the abundant blood vessels and VEGF. In addition, from the viewpoint of from developmental biology, the calvarial and tibial periostea are mainly responsible for intermembranous and endochondral ossification, respectively;4,6 these possibly contribute to the variations in the above-mentioned biomolecules (e.g., BMP and VEGF). In the current study, by using decellularization techniques to obtain biomimetic periosteum ECM scaffolds (DTP and DCP), we generated novel specific periosteum biomaterials for basic medicine research and clinical applications. To completely remove the cellular components and for good maintenance of bioactivity and hierarchical multilayer microstructure, we

combined freeze−thawing (breakdown of cell integrity and minimal microstructure damage), Triton X-100 and SDS treatments (disruption and dissolution of cell membranes), and DNase I treatment (digestion of nuclear and DNA fragments) for decellularization.33 Before decellularization of the calvarial periosteum, acetone was used to degrease the numerous adipose cells and avail decellularization reagents easily to permeate.19 The GAGs, which were present at high levels in the DTP and DCP, could act as “good biological macromolecules” for preserving various cell-growth factors (angiogenesis- and osteogenesis-related factors) in the ECM scaffolds.34 The GAG content of the calvarial periosteum was relatively low; low GAG content is believed to be closely related to lower Young modulus and higher stiffness.35 A suitable Young modulus is essential for osteogenic differentiation.36 VEGF is believed to be an essential factor for inducing angiogenesis and is also indirectly involved in the conversion of soft cartilaginous callus to bony callus during bone regeneration.37 In our study, the calvarial periosteum with higher VEGF content had vessels dispersed in the adipose layer. This structure may be closely related to bone formation in the case of the calvarial periosteum.38 On seeding HUVECs into two types of periosteum ECM scaffolds, DCP was found to show I


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Figure 9. Bone-formation-related gene expression in the DTP and DCP seeded with HUVECs and vessel formation related gene expression in the DTP and DCP seeded with MC-3T3 cells. (A−C) VEGF, HIF-1α, and ANG-1 expression of scaffolds and control group. (D−G) BMP-2, RUNX2, OSX, and collagen I expression of scaffolds and control group. ns, p > 0.05; *, p = 0.01−0.05; **, p = 0.001−0.01; ***, p < 0.001.

bone matrix,45 its angiogenic and osteogenic activities can be utilized under certain conditions of bone regeneration. We propose that periosteum ECM scaffolds (tibia- or calvariumderived scaffolds from animal or human donors) can be utilized, singly or in combination, as a “finished product” (similar to other decellularization scaffold products46) to enhance bone regeneration. For example, the DTP scaffold can be routinely used to promote repair of bone defects or injuries in adults. For children with incomplete ossification, the superior angiogenesis effect of DCP is beneficial for preventing possible future nonunion, malunion, or shortening of bones.

higher VEGF, HIF1-α, and Ang-I expression than DTP, indicating that it promoted angiogenesis. The HIF1-α and Ang-I can interplay with VEGF to regulate various cells (e.g., osteoblasts, endothelial cells, and fibroblasts) to enhance new vessel and bone formation.39,40 The BMP family of growth factors appear to be key molecules in the differentiation of periosteum progenitors during bone regeneration.41 The tibial periosteum contained more BMP-2 than the calvarial periosteum and the ALP expression was also correspondingly higher. In the nude mouse implantation model, osteogenic activities (BMP-2 content and calcifying nodules) were noted in the DTP and DCT groups with seeded MSCs. In the tibial periosteum scaffold, the osteogenic activities seemed to be more marked, which may be attributed to the direct effect of BMP on MSC osteoinduction and mineralization. In the in vitro cell study, on seeding MC3T3-E1 cells into ECM scaffolds, high BMP-2, collagen I, Runx2, and OSX expression was also found in DTP. These factors comprehensively promote endochondral and intramembranous ossification in bone regeneration.42−44 By contrast, relatively weak osteogenesis was found in the calvarial periosteum scaffold; this may be attributed to the fact that although it had lower BMP content, its higher angiogenic activity and ease in recellularization aided in osteogenesis. Although the periosteum scaffold has inferior biomechanics, lesser flexibility, and fewer sources compared to decellularized

5. CONCLUSIONS In this study, we developed decellularization protocols by using combinations of freeze−thawing and Triton-X 100, SDS, DNase I, and acetone treatments. With these protocols, cells could be completely removed from the tibial and calvarial periostea and hierarchical multilayer structures and components could be retained without cytotoxicity. Differences were found in the angiogenic and osteogenic effects of the DTP and DCP in CAM and nude mouse implantation models. Osteogenesis- and angiogenesis-related gene expression in cell-seeded (HUVECs and MC-3T3-E1 cells) tibial and calvarial periosteum ECM scaffolds was evaluated. Thus, this study provides specific site-derived periosteum biomaterials J


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with different functional properties, structures, and biomechanics for the different clinical requirements of bone regeneration.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.7b00548. Figure S1, decellularization treatment progress; Figure S2, uniaxial tensile stress−strain curves; Figure S3, bone and vessel formation related gene interactions (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (X.L.). *E-mail: [email protected] (P.C.). *E-mail: [email protected] (S.F.). ORCID

Xianfeng Lin: 0000-0003-1871-774X Author Contributions †

J.Z., Q.Z., and J.C. have contributed equally

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by the National Nature Science Fund of China (81702143, 81772387, and 81472064), Natural Science Fund of Zhejiang Province (Z15H060002), funds of science and technology department of Zhejiang Province (2009C03014-1), Zhejiang provincial program for the cultivation of high-level innovative health talents, Project of Health and Family Planning Commission of Zhejiang province (2015KYA133), and the Project of Education Department of Zhejiang Province (Y201017108).

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REFERENCES

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Preparation and Evaluation of Tibia- and Calvarium-Derived

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