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Journal of Peking University (Health Sciences) ›› 2021, Vol. 53 ›› Issue (4): 776-784. doi: 10.19723/j.issn.1671-167X.2021.04.026

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Biocompatibility and effect on bone formation of a native acellular porcine pericardium: Results of in vitro and in vivo

YOU Peng-yue,LIU Yu-hua(),WANG Xin-zhi,WANG Si-wen,TANG Lin   

  1. Department of Prosthodontics, Peking University School and Hospital of Stomatology & National Center of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology, Beijing 100081, China
  • Received:2021-06-07 Online:2021-08-18 Published:2021-08-25
  • Contact: Yu-hua LIU E-mail:lyhdentist@163.com

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Abstract:

Objective: To examine the morphology and biocompatibility of a native acellular porcine pericardium (APP) in vitro and to evaluate its barrier function and effects on osteogenesis when used in guided bone regeneration (GBR) in vivo. Methods: First, the morphology of APP (BonanGen®) was detected using a scanning electron microscope (SEM). Next, for biocompatibility test, proliferation of human bone marrow mesenchymal stem cells (hBMSCs) were determined using cell counting kit-8 (CCK-8) after being seeded 1, 3 and 7 days. Meanwhile, the cells stained with phalloidine and 4,6-diamidino-2-phenylindole (DAPI) were observed using a confocal laser scanning microscopy (CLSM) to view the morphology of cell adhesion and pattern of cell proliferation on day 5. A 3-Beagle dog model with 18 teeth extraction sockets was used for the further research in vivo. These sites were randomly treated by 3 patterns below: filled with Bio-Oss® and coverd by APP membrane (APP group), filled with Bio-Oss® and covered by Bio-Gide® membrane (BG group) and natural healing (blank group). Micro-CT and hematoxylin-eosin (HE) were performed after 4 and 12 weeks. Results: A bilayer and three-dimensional porous ultrastructure was identified for APP through SEM. In vitro, APP facilitated proliferation and adhesion of hBMSCs, especially after 7 days (P<0.05). In vivo, for the analysis of the whole socket healing, no distinct difference of new bone ratio was found between all the three groups after 4 weeks (P>0.05), however significantly more new bone regeneration was detected in APP group and BG group in comparison to blank group after 12 weeks (P<0.05). The radio of bone formation below the membrane was significantly higher in APP group and BG group than blank group after 4 and 12 weeks (P<0.05), however, the difference between APP group and BG group was merely significant in 12 weeks (P<0.05). Besides, less resorption of buccal crest after 4 weeks and 12 weeks was observed in APP group of a significant difference compared in blank group (P<0.05). The resorption in BG group was slightly lower than blank group (P>0.05). Conclusion: APP showed considerable biocompatibility and three-dimentional structure. Performing well as a barrier membrane in the dog alveolar ridge preservation model,APP significantly promoted bone regeneration below it and reduced buccal crest resorption. On the basis of this study, APP is a potential osteoconductive and osteoinductive biomaterial.

Key words: Pericardium, Resorbable membrane, Human bone marrow mesenchymal stem cell, Guided bone regeneration

CLC Number: 

  • R783.1

Figure 1

Choosing method of ROI and measurement of the vertical distance between the buccal and lingual(palatal) crest A, coronal view of region of interest (ROI); B, saggital view of ROI; C, central vertical line (CVL). BC, buccal crest; LC, lingual crest; VD, vertical distance."

Figure 2

Microstructure of APP (×300) A, smooth side of acellular porcine pericardium (APP); B, rough side of APP; C, cross section of APP."

Figure 3

CLSM view of the morphology of hBMSCs cultured on BG or APP membranes The left three rows were viewed at 250 magnification and the right three were at 5 000 magnification. Actins of cells were shown in green and nuclei were blue. APP, acellular porcine pericardium; BG, Bio-Gide."

Table 1

Percentage of different areas in ROI-1 at 4 weeks and 12 weeks /%"

Areas Time APP group BG group Blank group
Bone area 4 weeks 24.05±1.05 26.48±2.42 29.81±5.46
12 weeks 44.05±5.74c 41.50±6.22c 21.55±1.56ab
Bone marrow area 4 weeks 48.52±3.16 47.78±0.55 40.46±4.07ab
12 weeks 32.97±2.22c 33.06±4.63c 42.56±2.88ab
Soft tissue area 4 weeks 14.15±2.57 13.53±1.51 31.12±4.75ab
12 weeks 8.93±1.56c 12.56±3.45 35.88±3.03ab
Material area 4 weeks 13.24±3.07 13.58±2.42 -
12 weeks 14.46±3.91 13.49±1.93 -

Table 2

Percentage of different areas in ROI-2 at 4 and 12 weeks /%"

Areas Time APP group BG group Blank group
Bone area 4 weeks 31.26±2.76 30.36±1.72 11.26±0.91ab
12 weeks 42.56±2.88bc 37.62±1.87ac 15.09±1.81abc
Bone marrow area 4 weeks 30.05±1.70 30.59±1.20 10.67±0.71ab
12 weeks 25.69±1.96c 28.17±2.87 10.57±1.88ab
Soft tissue area 4 weeks 27.36±2.57 27.98±3.63 78.07±8.54ab
12 weeks 20.35±4.29 22.37±2.69 74.33±1.74ab
Material area 4 weeks 11.30±1.50 10.74±2.84 -
12 weeks 11.39±2.21 11.83±2.72 -

Table 3

Vertical distance of buccal and lingual crest /mm"

Time APP group BG group Blank group
4 weeks 0.89±0.09 1.32±0.33 1.53±0.29a
12 weeks 1.14±0.20 1.51±0.33 1.78±0.39a

Figure 4

Histological observations of extraction sockets healing after 4 weeks or 12 weeks APP group after 4 weeks’ healing (A, ×40; D, ×200), after 12 weeks’ healing (G, ×40; J, ×200); BG group after 4 weeks’ healing (B, ×40; E, ×200), after 12 weeks’ healing (H, ×40; K, ×200); BLANK group after 4 weeks’ healing (C, ×40; F, ×200); after 12 weeks’ healing (I, ×40; L, ×200). Asterisk, graft particles; NB, new bone; OB, old bone; blue arrow, osteoblast grew into the graft particles; black arrow, osteoclast; green arrow, osteoid."

Figure 5

Histological observations of coronal part of extraction sockets after healing of 4 weeks or 12 weeks APP group after 4 weeks’ healing (A, ×40; D, ×200), after 12 weeks’ healing (G, ×40; J, ×200); BG group after 4 weeks’ healing (B, ×40; E, ×200), after 12 weeks’ healing (H, ×40; K, ×200); blank group after 4 weeks’ healing (C, ×40; F, ×200), after 12 weeks’ healing (I, ×40; L, ×200). Asterisk, graft particles; NB, new bone; OB, old bone; blue arrow, osteoblast grew into the graft particles; green arrow, osteoid; M, membrane."

Figure 6

Schematic diagram of ROI area A, alveolar socket after tooth extraction; B, blank group after healing, soft tissue grew in and vertical bone lost; C, APP group or BG group after healing, less soft tissue grew in and less vertical bone lost."

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