Benefits of biphasic calcium phosphate hybrid scaffold-driven osteogenic differentiation of mesenchymal stem cells through upregulated leptin receptor expression

Journal of Orthopaedic Surgery and Research

Hsiao-Yang Cheng¹  Po-Han Chen³  Li-Jen Yuan⁴  Chao-Jan Wang¹  Chuen-Yung Yang³  Lih-Huei Chen⁴  Shih-Jung Liu²  Wen-Jer Chen⁴  Song-Shu Lin³  Chi-Chien Niu⁴ 

[1] Department of Radiology, Chang Gung Memorial Hospital, Taoyuan, Taiwan;Department of Mechanical Engineering, Chang Gung University, Taoyuan, Taiwan;Department of Orthopaedics, Chang Gung Memorial Hospital, No. 5, Fu-Hsing Street 333, Kweishan, Taoyuan, Taiwan;College of Medicine, Chang Gung University, Taoyuan, Taiwan

关键词: Leptin receptor;    Osteogenesis;    MSCs;    BCP;    Coralline HA;   

Others  :  1220950
DOI  :  10.1186/s13018-015-0236-2

 received in 2015-03-17, accepted in 2015-06-12,  发布年份 2015

【 摘 要 】

Background

The use of mesenchymal stem cells (MSCs) and coralline hydroxyapatite (HA) or biphasic calcium phosphate (BCP) as a bone substitute for posterolateral spinal fusion has been reported. However, the genes and molecular signals by which MSCs interact with their surrounding environment require further elucidation.

Methods

MSCs were harvested from bone grafting patients and identified by flow cytometry. A composite scaffold was developed using poly(lactide-co-glycolide) (PLGA) copolymer, coralline HA, BCP, and collagen as a carrier matrix for MSCs. The gene expression profiles of MSCs cultured in the scaffolds were measured by microarrays. The alkaline phosphatase (ALP) activity of the MSCs was assessed, and the expression of osteogenic genes and proteins was determined by quantitative polymerase chain reaction (Q-PCR) and Western blotting. Furthermore, we cultured rabbit MSCs in BCP or coralline HA hybrid scaffolds and transplanted these mixtures into rabbits for spinal fusion. We investigated the differences between BCP and coralline HA hybrid scaffolds by dual-energy X-ray absorptiometry (DEXA) and computed tomography (CT).

Results

Tested in vitro, the cells were negative for hematopoietic cell markers and positive for MSC markers. There was higher expression of 80 genes and lower of 101 genes of MSCs cultured in BCP hybrid scaffolds. Some of these genes have been shown to play a role in osteogenesis of MSCs. In addition, MSCs cultured in BCP hybrid scaffolds produced more messenger RNA (mRNA) for osteopontin, osteocalcin, Runx2, and leptin receptor (leptin-R) than those cultured in coralline HA hybrid scaffolds. Western blotting showed more Runx2 and leptin-R protein expression in BCP hybrid scaffolds. For in vivo results, 3D reconstructed CT images showed continuous bone bridges and fusion mass incorporated with the transverse processes. Bone mineral content (BMC) values were higher in the BCP hybrid scaffold group than in the coralline HA hybrid scaffold group.

Conclusions

The BCP hybrid scaffold for osteogenesis of MSCs is better than the coralline HA hybrid scaffold by upregulating expression of leptin-R. This was consistent with in vivo data, which indicated that BCP hybrid scaffolds induced more bone formation in a spinal fusion model.

【 授权许可】

   
2015 Niu et al.

【 图 表 】

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【 参考文献 】

• [1]Fernyhough JC, Schimandle JH, Weigel MC, Edwards CC, Levine AM. Chronic donor site pain complicating bone graft harvesting from the posterior iliac crest for spinal fusion. Spine. 1992; 17:1474-80.
• [2]Artzi Z, Weinreb M, Carmeli G, Lev-Dor R, Dard M, Nemcovsky CE. Histomorphometric assessment of bone formation in sinus augmentation utilizing a combination of autogenous and hydroxyapatite/biphasic tricalcium phosphate graft materials: at 6 and 9 months in humans. Clin Oral Implants Res. 2008; 19:686-92.
• [3]Yuan H, Fernandes H, Habibovic P, de Boer J, Barradas AMC, de Ruiter A et al.. Osteoinductive ceramics as a synthetic alternative to autologous bone grafting. PNAS. 2010; 107:13614-9.
• [4]Yang C, Unursaikhan O, Lee JS, Jung UW, Kim CS, Choi SH. Osteoconductivity and biodegradation of synthetic bone substitutes with different tricalcium phosphate contents in rabbits. J Biomed Mater Res Part B. 2014; 102B:80-8.
• [5]Lin SS, Ueng SWN, Niu CC, Yuan LJ, Yang CY, Chen WJ et al.. Hyperbaric oxygen promotes osteogenic differentiation of bone marrow stromal cells by regulating Wnt3a/β-catenin signaling—an in vitro and in vivo study. Stem Cell Res. 2014; 12:260-74.
• [6]Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD et al.. Multilineage potential of adult human mesenchymal stem cells. Science. 1999; 284:143-7.
• [7]Yoshikawa T, Ohgishi H, Tamai S. Immediate bone forming capability of prefabricated osteogenic hydroxyapatite. J Biomed Mater Res. 1996; 32:481-92.
• [8]Livingston Arinzeh T, Peter S, Archambault M, Van Den Bos C, Gordon S et al.. Allogeneic mesenchymal stem cells regenerate bone in a critical-sized canine segmental defect. J Bone Joint Surg Am. 2003; 85A:1927-35.
• [9]Huang JW, Lin SSMS, Chen LH, Liu SJ, Niu CC, Yuan LJ et al.. The use of fluorescence-labeled mesenchymal stem cells in poly(lactide-co-glycolide)/hydroxyapatite/collagen hybrid graft as a bone substitute for posterolateral spinal fusion. J Trauma. 2011; 70:1495-502.
• [10]Kai T, Guo SQ, Dang GT. In vivo evaluation of bone marrow stromal-derived osteoblasts-porous calcium phosphate ceramic composites as bone graft substitute for lumbar intervertebral spinal fusion. Spine. 2003; 28:1653-8.
• [11]Chen G, Deng C, Li YP. TGF-beta and BMP signaling in osteoblast differentiation and bone formation. Int J Biol Sci. 2012; 8:272-88.
• [12]St-Jacques B, Hammerschmidt M, McMahon AP. Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev. 1999; 13:2072-86.
• [13]Hilton MJ, Tu X, Wu X, Bai S, Zhao H, Kobayashi T et al.. Notch signaling maintains bone marrow mesenchymal progenitors by suppressing osteoblast differentiation. Nat Med. 2008; 14:306-14.
• [14]Lin GL, Hankenson KD. Integration of BMP, Wnt, and notch signaling pathways in osteoblast differentiation. J Cell Biochem. 2011; 112:3491-501.
• [15]Ornitz DM, Marie PJ. FGF signaling pathways in endochondral and intramembranous bone development and human genetic disease. Genes Dev. 2002; 16:1446-65.
• [16]Takikawa S, Bauer TW, Kambic H, Togawa D. Comparative evaluation of the osteoinductivity of two formulations of human demineralized bone matrix. J Biomed Mater Res A. 2003; 65:37-42.
• [17]Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature. 1994; 372:425-32.
• [18]Reseland JE, Syversen U, Bakke I, Qvigstad G, Eide LG, Hjertner O et al.. Leptin is expressed in and secreted from primary cultures of human osteoblasts and promotes bone mineralization. J Bone Miner Res. 2001; 16:1426-33.
• [19]Noh M. Interleukin-17A increases leptin production in human bone marrow mesenchymal stem cells. Biochem Pharmacol. 2012; 83:661-70.
• [20]Hess R, Pino AM, Rios S et al.. High affinity leptin receptors are present in human mesenchymal stem cells (MSCs) derived from control and osteoporotic donors. J Cell Biochem. 2005; 94:50-7.
• [21]Astudillo P, Rios S, Pastenes L, Pino AM, Rodriguez JP. Increased adipogenesis of osteoporotic human-mesenchymal stem cells (MSCs) characterizes by impaired leptin action. J Cell Biochem. 2008; 103:1054-65.
• [22]Rodriguez JP, Astudillo P, Rios S et al.. Involvement of adipogenic potential of human bone marrow mesenchymal stem cells (MSCs) in osteoporosis. Curr Stem Cell Res Ther. 2008; 3:208-18.
• [23]Xu JC, Wu GL, Liu HL et al.. The effect of leptin on the osteoinductive activity of recombinant human bone morphogenetic protein-2 in nude mice. Saudi Med J. 2010; 31:615-21.
• [24]Holloway WR, Coller FM, Aitken CJ et al.. Leptin inhibits osteoclast generation. J Bone Miner Res. 2002; 17:200-9.
• [25]Xynos ID, Edgar AJ, Buttery LDK, Hench LL, Polak JM. Gene-expression profiling of human osteoblasts following treatment with the ionic products of Bioglass 45S5 dissolution. J Biomed Mater Res. 2001; 55:151-7.
• [26]Granjeaud S, Bertucci F, Jordan BR. Expression profiling: DNA arrays in many guises. BioEssays. 1999; 21:781-90.
• [27]Barradas A, Yuan H, Blitterswijk CA, Habibovic P. Osteoinductive biomaterials: current knowledge of properties, experimental models and biological mechanisms. Eur Cells Mater. 2011; 21:407-29.
• [28]Barbieri D, Yuan H, de Groot F, Walsh WR, de Bruijn JD. Influence of different polymeric gels on the ectopic bone forming ability of an osteoinductive biphasic calcium phosphate ceramic. Acta Biomater. 2011; 7:2007-14.
• [29]Tan YF, Wang G, Fan HS, Wang XL, Lu J, Zhang XD. Expression of core binding factor 1 and osteoblastic markers in C2C12 cells induced by calcium phosphate ceramics in vitro. J Biomed Mater Res Part A. 2007; 82:152-9.
• [30]Muller P, Bulnheim U, Diener A, Luthen F, Teller M, Klinkenberg ED et al.. Calcium phosphate surfaces promote osteogenic differentiation of mesenchymal stem cells. J Cell Mol Med. 2008; 12:281-91.
• [31]Yuan H, Yang Z, Li Y, Zhang X, De Bruijn JD, De Groot K. Osteoinduction by calcium phosphate biomaterials. J Mater Sci Mater Med. 1998; 9:723-6.
• [32]Barradas AM, Monticone V, Hulsman M, Danoux C, Fernandes H, Tahmasebi Birgani Z et al.. Molecular mechanisms of biomaterial-driven osteogenic differentiation in human mesenchymal stromal cells. Integr Biol. 2013; 5:920-31.
• [33]Long F. Building strong bones: molecular regulation of the osteoblast lineage. Nat Rev Mol Cell Biol. 2012; 13:27-38.
• [34]Deschaseaux F, Sensébé L, Heymann D. Mechanisms of bone repair and regeneration. Trends Mol Med. 2009; 15:417-29.
• [35]Tang Z, Wang Z, Qing F, Ni Y, Fan Y, Tan Y et al.. Bone morphogenetic protein Smads signaling in mesenchymal stem cells affected by osteoinductive calcium phosphate ceramics. J Biomed Mater Res Part A. 2015; 103:1001-10.
• [36]Grundberg E, Brändström H, Lam KCL, Gurd S, Ge B, Harmsen E et al.. Systematic assessment of the human osteoblast transcriptome in resting and induced primary cells. Physiol Genomics. 2008; 33:301-11.
• [37]Ghilardi N, Ziegler S, Wiestner A, Stoffel R, Heim MH, Skoda RC. Defective STAT signaling by the leptin receptor in diabetic mice. PNAS. 1996; 93:6231-5.
• [38]Dumond H, Presle N, Terlain B, Mainard D, Loeuille D, Netter P et al.. Evidence for a key role of leptin in osteoarthritis. Arthritis Rheum. 2003; 48:3118-29.
• [39]Welt CK, Chan JL, Bullen J, Murphy R, Smith P, DePaoli AM et al.. Recombinant human leptin in women with hypothalamic amenorrhea. N Engl J Med. 2004; 351:987-97.
• [40]Baas J, Svaneby D, Jensen TB, Elmengaard B, Bechtold J, Soballe K. Coralline hydroxyapatite granules inferior to morselized allograft around uncemented porous Ti implants: unchanged fixation by addition of concentrated autologous bone marrow aspirate. J Biomed Mater Res A. 2011; 99:9-15.
• [41]Boden SD, Martin GJ, Morone M, Ugbo JL, Titus L, Hutton WC. The use of coralline hydroxyapatite with bone marrow, autogenous bone graft, or osteoinductive bone protein extract for posterolateral lumbar spine fusion. Spine. 1999; 24:320-7.
• [42]Nickoli MS. Ceramic-based bone grafts as a bone grafts extender for lumbar spine arthrodesis: a systematic review. Global Spine J. 2014; 4:211-6.
• [43]Arinzeh TL, Tran T, Mcalary J, Daculsi G. A comparative study of biphasic calcium phosphate ceramics for human mesenchymal stem-cell-induced bone formation. Biomaterials. 2005; 26:3631-8.
• [44]Siggers K, Frei H, Fernlund G, Rossi F. Effect of bone graft substitute on marrow stromal cell proliferation and differentiation. J Biomed Mater Res A. 2010; 94:877-85.
• [45]Dorozhkin SV. Calcium orthophosphate-based bioceramics. Materials. 2013; 6:3840-942.
• [46]Draenert M, Draenert A, Draenert K. Osseointegration of hydroxyapatite and remodeling-resorption of tricalciumphosphate ceramics. Microsc Res Tech. 2013; 76:370-80.
• [47]Tancred DC, McCormack BA, Carr AJ. A synthetic bone implant macroscopically identical to cancellous bone. Biomaterials. 1998; 19:2303-11.
• [48]Schliephake H, Neukam FW, Klosa D. Influence of pore dimensions on bone ingrowth into porous hydroxylapatite blocks used as bone graft substitutes. A histometric study. Int J Oral Maxillofac Surg. 1991; 20:53-8.
• [49]Vani R, Girija EK, Elayaraja K, Parthiban PS, Kesavamoorthy R, Narayana KS. Hydrothermal synthesis of porous triphasic hydroxyapatite/(α and β) tricalcium phosphate. J Mater Sci Mater Med. 2009; 20:S43-8.
• [50]Yuan H, van Blitterswijk CA, de Groot K, de Bruijn JD. A comparison of bone formation in biphasic calcium phosphate (BCP) and hydroxyapatite (HA) implanted in muscle and bone of dogs at different time periods. J Biomed Mater Res. 2006; 78A:139-47.
• [51]Habibovic P, Yuan H, van der Valk CM, Meijer G, van Blitterswijk CA, de Groot K. 3D microenvironment as essential element for osteoinduction by biomaterials. Biomaterials. 2005; 26:3565-75.
• [52]Liu C, Batliwalla F, Li W, Lee A, Roubenoff R, Beckman E et al.. Genome-wide association scan identifies candidate polymorphisms associated with differential response to anti-TNF treatment in rheumatoid arthritis. Mol Med. 2008; 14:575-81.
• [53]White CD, Brown MD, Sacks DB. IQGAPs in cancer: a family of scaffold proteins underlying tumorigenesis. FEBS Lett. 2009; 583:1817-24.
• [54]Tseng S, Reddi AH, Di Cesare PE. Cartilage oligomeric matrix protein (COMP): a biomarker of arthritis. Biomark Insights. 2009; 4:33-44.