Healing of Bone Defects in Pig's Femur Using Mesenchymal Cells Originated from the Sinus Membrane with Different Scaffolds

Rita Bou Assaf, Kazem Zibara, Mohammad Fayyad-Kazan, Fatima Al-Nemer, Manal Cordahi, Saad Khairallah, Bassam Badran, Antoine Berbéri, Rita Bou Assaf, Kazem Zibara, Mohammad Fayyad-Kazan, Fatima Al-Nemer, Manal Cordahi, Saad Khairallah, Bassam Badran, Antoine Berbéri

Abstract

Objective: Repairing bone defects, especially in older individuals with limited regenerative capacity, is still a big challenge. The use of biomimetic materials that can enhance the restoration of bone structure represents a promising clinical approach. In this study, we evaluated ectopic bone formation after the transplantation of human maxillary Schneiderian sinus membrane- (hMSSM-) derived cells embedded within various scaffolds in the femur of pigs.

Methods: The scaffolds used were collagen, gelatin, and hydroxyapatite/tricalcium phosphate (HA/βTCP) where fibrin/thrombin was used as a control. Histological analysis was performed for the new bone formation. Quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC) were used to assess mRNA and protein levels of specific osteoblastic markers, respectively.

Results: Histological analysis showed that the three scaffolds we used can support new bone formation with a more pronounced effect observed in the case of the gelatin scaffold. In addition, mRNA levels of the different tested osteoblastic markers Runt-Related Transcription Factor 2 (RUNX-2), osteonectin (ON), osteocalcin (OCN), osteopontin (OPN), alkaline phosphatase (ALP), and type 1 collagen (COL1) were higher, after 2 and 4 weeks, in cell-embedded scaffolds than in control cells seeded within the fibrin/thrombin scaffold. Moreover, there was a very clear and differential expression of RUNX-2, OCN, and vimentin in osteocytes, osteoblasts, hMSSM-derived cells, and bone matrix. Interestingly, the osteogenic markers were more abundant, at both time points, in cell-embedded gelatin scaffold than in other scaffolds (collagen, HA/βTCP, fibrin/thrombin).

Conclusions: These results hold promise for the development of successful bone regeneration techniques using different scaffolds embedded with hMSSM-derived cells. This trial is registered with NCT02676921.

Conflict of interest statement

The authors declare that there is no conflict of interest regarding the publication of this article.

Copyright © 2019 Rita Bou Assaf et al.

Figures

Figure 1
Figure 1
The eight defects were divided into two groups depending on the type of scaffolds and cells that they have received. a1: mesenchymal sinus membrane cell with collagen. b1: mesenchymal sinus membrane cell with gelatin (hemostatic sponge). c1: mesenchymal sinus membrane cell with βTCP and HA. d1: mesenchymal sinus membrane cell with fibrin and thrombin. a2: collagen without stem cells. b2: gelatin without stem cells. c2: βTCP and HA without stem cells. d2: fibrin and thrombin.
Figure 2
Figure 2
Hematoxylin and eosin staining of histological micrographs from paraffin-embedded scaffolds implanted in the femur of pigs at 2 and 4 weeks. Asterisks (∗) represent the new bone formation and F corresponds to the fibroblastic reaction while I represents the inflammatory reactions. New bone formation was detected in groups implanted with scaffolds along with the hMSSM cells, in comparison with the control group with scaffolds alone. Magnification is 40x.
Figure 3
Figure 3
Hematoxylin and eosin staining of histological micrographs from paraffin-embedded scaffolds implanted in the femur of pigs at 6 and 8 weeks. Asterisks (∗) represent the mature bone in the inner and outer areas of the scaffolds. Note the presence of a chronic inflammatory exudate within the sections. Magnification is 40x.
Figure 4
Figure 4
Quantitative real-time PCR (qRT-PCR) of different osteoblastic markers. (a) RUNX-2, (b) ON, (c) OCN, (d) OPN, (e) ALP, and (f) COL1 mRNA levels in cell-embedded scaffolds from different implants at 2 or 4 weeks. The expression levels are relative to those obtained in cells+fibrin-thrombin (control). Data were normalized to GAPDH levels. Each value represents a mean ± SEM for three independent experiments (n = 3). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001vs. cells with gelatin scaffold (Student's t-test).
Figure 5
Figure 5
Immunohistochemistry (IHC) of RUNX-2 osteoblastic marker. Expression of RUNX-2 protein in scaffold-embedded hMSSM-derived cells from different implants after 2 or 4 weeks. Asterisks (∗) represent the new bone formation while solid arrows (→) correspond to the positively stained osteoblast cells.
Figure 6
Figure 6
Immunohistochemistry (IHC) of OCN osteoblastic marker. Expression of OCN protein in scaffold-embedded hMSSM-derived cells from different implants after 2 or 4 weeks. Staining showed a very clear differential expression of OCN in osteocytes, osteoblasts, hMSSM-derived cells, and bone matrix. Interestingly, OCN was more abundant, at both time points, in gelatin scaffold-embedded cells than the other scaffolds (collagen, HA/βTCP, and fibrin/thrombin). Asterisks (∗) represent the new bone matrix formation and regular arrows (→) correspond to the positively stained osteoblast cells while bold arrows (•→) correspond to the positively stained osteocytes.
Figure 7
Figure 7
Immunohistochemistry (IHC) of vimentin osteoblastic marker. Expression of vimentin protein in scaffold-embedded hMSSM-derived cells from different implants after 2 or 4 weeks. Staining showed a very clear differential expression of vimentin in osteocytes, osteoblasts, hMSSM-derived cells, and bone matrix. Interestingly, vimentin was more abundant, at both time points, in gelatin scaffold-embedded cells than the other scaffolds (collagen, HA/βTCP, and fibrin/thrombin). Asterisks (∗) represent the new bone matrix formation and solid short arrows correspond to the positively stained osteoblast cells while solid long arrows correspond to the positively stained osteocytes.

References

    1. Myeroff C., Archdeacon M. Autogenous bone graft: donor sites and techniques. The Journal of Bone and Joint Surgery-American Volume. 2011;93(23):2227–2236. doi: 10.2106/JBJS.J.01513.
    1. Ebraheim N. A., Elgafy H., Xu R. Bone-graft harvesting from iliac and fibular donor sites: techniques and complications. Journal of the American Academy of Orthopaedic Surgeons. 2001;9(3):210–218. doi: 10.5435/00124635-200105000-00007.
    1. Joyce M. J. Safety and FDA regulations for musculoskeletal allografts: perspective of an orthopaedic surgeon. Clinical Orthopaedics and Related Research. 2005;435:22–30. doi: 10.1097/01.blo.0000165849.32661.5e.
    1. Amini A. R., Laurencin C. T., Nukavarapu S. P. Bone tissue engineering: recent advances and challenges. Critical Reviews in Biomedical Engineering. 2012;40(5):363–408. doi: 10.1615/CritRevBiomedEng.v40.i5.10.
    1. Roseti L., Parisi V., Petretta M., et al. Scaffolds for bone tissue engineering: state of the art and new perspectives. Materials Science and Engineering: C. 2017;78:1246–1262. doi: 10.1016/J.MSEC.2017.05.017.
    1. Laurencin C., Khan Y., El-Amin S. F. Bone graft substitutes. Expert Review of Medical Devices. 2014;3(1):49–57. doi: 10.1586/17434440.3.1.49.
    1. Polo-Corrales L., Latorre-Esteves M., Ramirez-Vick J. E. Scaffold design for bone regeneration. Journal of Nanoscience and Nanotechnology. 2014;14(1):15–56. doi: 10.1166/jnn.2014.9127.
    1. Jafarian M., Eslaminejad M. B., Khojasteh A., et al. Marrow-derived mesenchymal stem cells-directed bone regeneration in the dog mandible: a comparison between biphasic calcium phosphate and natural bone mineral. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology. 2008;105(5):e14–e24. doi: 10.1016/j.tripleo.2008.01.010.
    1. Khojasteh A., Eslaminejad M. B., Nazarian H., et al. Vertical bone augmentation with simultaneous implant placement using particulate mineralized bone and mesenchymal stem cells: a preliminary study in rabbit. The Journal of Oral Implantology. 2013;39(1):3–13. doi: 10.1563/AAID-JOI-D-10-00206.
    1. Khojasteh A., Behnia H., Hosseini F. S., Dehghan M. M., Abbasnia P., Abbas F. M. The effect of PCL-TCP scaffold loaded with mesenchymal stem cells on vertical bone augmentation in dog mandible: a preliminary report. Journal of Biomedical Materials Research Part B: Applied Biomaterials. 2013;101B(5):848–854. doi: 10.1002/jbm.b.32889.
    1. Yang M., Ma Q. J., Dang G. T., Ma K. T., Chen P., Zhou C. Y. In vitro and in vivo induction of bone formation based on ex vivo gene therapy using rat adipose-derived adult stem cells expressing BMP-7. Cytotherapy. 2005;7(3):273–281. doi: 10.1080/14653240510027244.
    1. Peterson B., Zhang J., Iglesias R., et al. Healing of critically sized femoral defects, using genetically modified mesenchymal stem cells from human adipose tissue. Tissue Engineering. 2005;11(1-2):120–129. doi: 10.1089/ten.2005.11.120.
    1. Huang G. T.-J., Gronthos S., Shi S. Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine. J Dent Res. 2009;88(9):792–806. doi: 10.1177/0022034509340867.
    1. Morad G., Kheiri L., Khojasteh A. Dental pulp stem cells for in vivo bone regeneration: a systematic review of literature. Archives of Oral Biology. 2013;58(12):1818–1827. doi: 10.1016/j.archoralbio.2013.08.011.
    1. Houshmand B., Behnia H., Khoshzaban A., et al. Osteoblastic differentiation of human stem cells derived from bone marrow and periodontal ligament under the effect of enamel matrix derivative and transforming growth factor-beta. The International Journal of Oral & Maxillofacial Implants. 2013;28(6):e440–e450. doi: 10.11607/jomi.te24.
    1. Srouji S., Ben-David D., Lotan R., Riminucci M., Livne E., Bianco P. The innate osteogenic potential of the maxillary sinus (Schneiderian) membrane: an ectopic tissue transplant model simulating sinus lifting. International Journal of Oral and Maxillofacial Surgery. 2010;39(8):793–801. doi: 10.1016/j.ijom.2010.03.009.
    1. Berbéri A., Al-Nemer F., Hamade E., Noujeim Z., Badran B., Zibara K. Mesenchymal stem cells with osteogenic potential in human maxillary sinus membrane: an in vitro study. Clinical Oral Investigations. 2017;21(5):1599–1609. doi: 10.1007/s00784-016-1945-6.
    1. Stoppel W. L., Ghezzi C. E., McNamara S. L., III L. D. B., Kaplan D. L. Clinical applications of naturally derived biopolymer-based scaffolds for regenerative medicine. Annals of Biomedical Engineering. 2015;43(3):657–680. doi: 10.1007/s10439-014-1206-2.
    1. Khojasteh A., Behnia H., Dashti S. G., Stevens M. Current trends in mesenchymal stem cell application in bone augmentation: a review of the literature. Journal of Oral and Maxillofacial Surgery. 2012;70(4):972–982. doi: 10.1016/j.joms.2011.02.133.
    1. Bou Assaf R., Fayyad-Kazan M., al-Nemer F., et al. Evaluation of the osteogenic potential of different scaffolds embedded with human stem cells originated from Schneiderian membrane: an in vitro study. BioMed Research International. 2019;2019:10. doi: 10.1155/2019/2868673.2868673
    1. O'Brien F. J. Biomaterials & scaffolds for tissue engineering. Materials Today. 2011;14(3):88–95. doi: 10.1016/S1369-7021(11)70058-X.
    1. Bose S., Roy M., Bandyopadhyay A. Recent advances in bone tissue engineering scaffolds. Trends in Biotechnology. 2012;30(10):546–554. doi: 10.1016/j.tibtech.2012.07.005.
    1. Dong C., Lv Y. Application of collagen scaffold in tissue engineering: recent advances and new perspectives. Polymers. 2016;8(2):p. 42. doi: 10.3390/polym8020042.
    1. Meinel L., Karageorgiou V., Fajardo R., et al. Bone tissue engineering using human mesenchymal stem cells: effects of scaffold material and medium flow. Annals of Biomedical Engineering. 2004;32(1):112–122. doi: 10.1023/B:ABME.0000007796.48329.b4.
    1. Kuo Z.-K., Lai P. L., Toh E. K. W., et al. Osteogenic differentiation of preosteoblasts on a hemostatic gelatin sponge. Scientific Reports. 2016;6(1, article 32884) doi: 10.1038/srep32884.
    1. Paganelli C., Fontana P., Porta F., Majorana A., Pazzaglia U. E., Sapelli P. L. Indications on suitable scaffold as carrier of stem cells in the alveoloplasty of cleft palate. Journal of Oral Rehabilitation. 2006;33(8):625–629. doi: 10.1111/j.1365-2842.2005.01594.x.
    1. Cegielski M., Dziewiszek W., Zabel M., et al. Experimental xenoimplantation of antlerogenic cells into mandibular bone lesions in rabbits: two-year follow-up. In Vivo. 2010;24(2):165–172.
    1. Arias-Gallo J., Chamorro-Pons M., Avendaño C., Giménez-Gallego G. Influence of acidic fibroblast growth factor on bone regeneration in experimental cranial defects using spongostan and Bio-Oss as protein carriers. The Journal of Craniofacial Surgery. 2013;24(5):1507–1514. doi: 10.1097/SCS.0b013e31828f2469.
    1. Srouji S., Ben-David D., Funari A., Riminucci M., Bianco P. Evaluation of the osteoconductive potential of bone substitutes embedded with schneiderian membrane- or maxillary bone marrow-derived osteoprogenitor cells. Clinical Oral Implants Research. 2013;24(12):1288–1294. doi: 10.1111/j.1600-0501.2012.02571.x.
    1. Thorwarth M., Schultze-Mosgau S., Kessler P., Wiltfang J., Schlegel K. A. Bone regeneration in osseous defects using a resorbable nanoparticular hydroxyapatite. Journal of Oral and Maxillofacial Surgery. 2005;63(11):1626–1633. doi: 10.1016/j.joms.2005.06.010.
    1. Rubessa M., Polkoff K., Bionaz M., et al. Use of pig as a model for mesenchymal stem cell therapies for bone regeneration. Animal Biotechnology. 2017;28(4):275–287. doi: 10.1080/10495398.2017.1279169.

Source: PubMed

Спонсоры и соавторы

Заболевания

Медикаментозные вмешательства

Подписаться