Bone marrow-derived mesenchymal stem cells assembled with low-dose BMP-2 in a three-dimensional hybrid construct enhances posterolateral spinal fusion in syngeneic rats.
BACKGROUND
The combination of potent osteoinductive growth factor, functional osteoblastic cells, and osteoconductive materials to induce bone formation is a well-established concept in bone tissue engineering. However, supraphysiological dose of growth factor, such as recombinant human bone morphogenetic protein 2 (rhBMP-2), which is necessary in contemporary clinical application, have been reported to result in severe side effects.


OBJECTIVE
We hypothesize that the synergistic osteoinductive capacity of low-dose bone morphogenetic protein 2 (BMP-2) combined with undifferentiated bone marrow-derived stromal cells (BMSCs) is comparable to that of osteogenically differentiated BMSCs when used in a rodent model of posterolateral spinal fusion.


METHODS
A prospective study using a rodent model of posterolateral spinal fusion was carried out.


METHODS
Thirty-six syngeneic Fischer rats comprised the patient sample.


METHODS
Six groups of implants were evaluated as follows (n=6): (1) 10 µg BMP-2 with undifferentiated BMSCs; (2) 10 µg BMP-2 with osteogenic-differentiated BMSCs; (3) 2.5 µg BMP-2 with undifferentiated BMSCs; (4) 2.5 µg BMP-2 with osteogenic-differentiated BMSCs; (5) 0.5 µg BMP-2 with undifferentiated BMSCs; and (6) 0.5 µg BMP-2 with osteogenic-differentiated BMSCs. Optimal in vitro osteogenic differentiation of BMSCs was determined by quantitative real-time polymerase chain reaction (qRT-PCR) gene analysis whereas in vivo bone formation capacity was evaluated by manual palpation, micro-computed tomography, and histology.


RESULTS
Rat BMSCs cultured in fibrin matrix that was loaded into the pores of medical-grade poly epsilon caprolactone tricalcium phosphate scaffolds differentiated toward osteogenic lineage by expressing osterix, runt-related transcription factor 2, and osteocalcium mRNA when supplemented with dexamethasone, ascorbic acid, and β-glycerophosphate. Whereas qRT-PCR revealed optimal increase in osteogenic genes expression after 7 days of in vitro culture, in vivo transplantation study showed that pre-differentiation of BMSCs before transplantation failed to promote posterolateral spinal fusion when co-delivered with low-dose BMP-2 (1/6 or 17% fusion rate). In contrast, combined delivery of undifferentiated BMSCs with low-dose BMP-2 (2.5 µg) demonstrated significantly higher fusion rate (4/6 or 67%) as well as significantly increased volume of new bone formation (p<.05).


CONCLUSIONS
In summary, this study supports the combination of undifferentiated BMSCs and low-dose rhBMP-2 for bone tissue engineering construct.
Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Level 11, NUHS Tower Block, 1E Kent Ridge Rd, 119228, Singapore.
Tao
T
Hu
Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Level 11, NUHS Tower Block, 1E Kent Ridge Rd, 119228, Singapore.
Sunny Akogwu
SA
Abbah
Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Level 11, NUHS Tower Block, 1E Kent Ridge Rd, 119228, Singapore.
Soo Yein
SY
Toh
Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Level 11, NUHS Tower Block, 1E Kent Ridge Rd, 119228, Singapore.
Ming
M
Wang
Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Level 11, NUHS Tower Block, 1E Kent Ridge Rd, 119228, Singapore.
Raymond Wing Moon
RW
Lam
Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Level 11, NUHS Tower Block, 1E Kent Ridge Rd, 119228, Singapore.
Mathanapriya
M
Naidu
Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, #06-06 Immunos, 138648, Singapore.
Gajadhar
G
Bhakta
Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Level 11, NUHS Tower Block, 1E Kent Ridge Rd, 119228, Singapore; Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, #06-06 Immunos, 138648, Singapore.
Simon M
SM
Cool
Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Level 11, NUHS Tower Block, 1E Kent Ridge Rd, 119228, Singapore; Singapore Bioimaging Consortium (SBIC), Agency for Science, Technology and Research (A*STAR), 11 Biopolis Way, #02-02 Helios Building, 138667, Singapore.
Kishore
K
Bhakoo
Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore, Block EA, #03-12, 9 Engineering Drive 1, 117575, Singapore.
Jun
J
Li
Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Level 11, NUHS Tower Block, 1E Kent Ridge Rd, 119228, Singapore; Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore, Block EA, #03-12, 9 Engineering Drive 1, 117575, Singapore.
James Cho-Hong
JC
Goh
Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Level 11, NUHS Tower Block, 1E Kent Ridge Rd, 119228, Singapore. Electronic address: doswhk@nus.edu.sg.
Hee-Kit
HK
Wong
D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000494:pharmacology*; D002478:Cells, Cultured; D006801:Humans; D008297:Male; D045164:Mesenchymal Stem Cell Transplantation / Q000379:methods; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects; D010012:Osteogenesis; D051381:Rats; D011916:Rats, Inbred F344; D011994:Recombinant Proteins / Q000494:pharmacology; D013123:Spinal Fusion / Q000379:methods*; D023822:Tissue Engineering / Q000379:methods; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D055396:Bone Morphogenetic Protein 2; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C499469:recombinant human bone morphogenetic protein-2
Bone tissue engineering; Differentiation; Growth factor therapy; Hybrid construct; Stem cell; Undifferentiation