Recombinant Human Bone Morphogenic Protein-2 and MSC for bone

Plain Language Summary of Work

This research centered on the investigation of the effects of Diabetes Mellitus (DM), local delivery of an exogenic genetic-protein (rhBMP-2), and stem cell augmentation on bone segmental defects healing in the presence of diabetes mellitus in a rat model. Our study is the first to analyze the effects of DM in a segmental defect model and treated with the application of a local rhBMP-2 delivery system. We demonstrated that animals treated with rhBMP-2/collagen carrier–treated had significantly increased bridging of the defects and increased radiographic scores and increased histomorphometric and mechanical testing parameters of new bone formation compared with the buffer/collagen-treated group in both DM and non-DM rats. This study demonstrated the effectiveness of rhBMP in the presence of DM in a segmental defect and provides insight in the potential role of rhBMP-2 in this difficult clinical application.

We investigated further to analyze the effect of MSC augmentation upon bone Allograft (Allo) incorporation, using histomorphometric (histological) outcome parameters upon a segmental defect model in the presence of diabetes mellitus (DM). Our study demonstrated that DM delayed Allo incorporation and bone formation in DM rats compared to that of its non-DM counterpart. Our results support the concept that MSC augmentation significantly increases bone formation in allograft incorporation in the presence of DM. The amount of bone increased greater than 300% from an average of (1.07 mm²) in DM/DBM animals to (3.57 mm²) in DM/MSC animals at the early time point of 4 weeks. Additionally, more mature bone was observed with local MSC augmentation in the DM animals at both 4 and 8 weeks. The current study confirms the potential of MSC augmentation upon allograft incorporation in a DM segmental defect model by demonstrating that a DM segmental defect treated with MSC is histomorphometrically indistinguishable from a non-DM segmental defect utilizing a current mainstay treatment of DBM alone. Our study is the first study exploring MSC as a potential adjuvant for allograft incorporation in a DM population.

Resulting Publications

Publications (Journal articles):

Vikrant Azad, Eric Breitbart, Loay Al-Zube, Sloane Yeh, Ann Marie Simon, J. Patrick O’Connor, Sheldon Lin. “rhBMP-2 enhances the bone healing response in a diabetic rat segmental defect model”. J Orthop Trauma. 2009 Apr; 23(4):267-76.
Eric A. Breitbart, Sharonda Meade, Vikrant Azad, Sloane Yeh, Loay Al-Zube, Yee-Shuan Lee, Joseph Benevenia, Treena Livingston-Arinzeh, Sheldon Lin. “Mesenchymal Stem Cells Accelerate Bone Allograft Incorporation in the Presence of Diabetes Mellitus”. J Orthop Res. 2010 July; 28(7): 942-949.
Jacobsen K.; Szczepanowski K.; Al-Zube L. A., Kim J., and Lin S. S., 2007. “The Role of Intraoperative Bone Marrow Aspirate Stem Cell Concentration as a Bone Grafting Technique”. Techniques in Foot & Ankle Surgery. 2008 June; 7(2):84-89.

Technical Summary of Work

This work involved the investigation of the effects of local recombinant human bone morphogenic protein-2 (rhBMP-2) delivery on bone healing in segmental femoral defects in the presence of diabetes mellitus. Using collagen sponge soaked with rhBMP-2 inserted in a mid-diaphyseal 3.0-mm femoral defect in rats fixed with polyimide plate and stainless steel screws, we demonstrated the effectiveness of rhBMP in increasing new bone formation in a segmental defect in the presence of diabetes Mellitus. Radiographic, histomorphometric, neoangiogenesis, and mechanical testing were employed. Radiographs assessed over a 6-point grading system showed statistically significant improvement in scores in rhBMP-2–treated rats at 6 weeks (P < 0.001). Histomorphometric analysis showed statistically significant increase in area of new bone formation between rats treated with rhBMP-2 at both 3 and 6 weeks (P < 0.001). On Platelet endothelial cell adhesion molecule-1 staining at 3 weeks, the mean number of vessels in rhBMP-2–treated DM rats was 12.76 ± 5.43/mm²compared with 4.49 ± 1.89/mm² in buffer (collagen sponge with saline) treated DM rats (P = 0.034). On mechanical testing, all 4 DM/buffer rats had nonunion. In DM/rhBMP-2 rats, the torque to failure and torsional rigidity values were 393.57±233.3 (P < 0.03) and 29,711± 6224 (P < 0.002), respectively. Further, our model also revealed that a single application of rhBMP-2 resulted in new bone formation in the DM group comparable to the non-DM group, thus demonstrating the ability of rhBMP-2 in negating the deleterious effects of DM on new bone formation.

We also explored the effects of Mesenchymal Stem Cells (MSC) augmentation upon bone allograft (Allo) incorporation in the presence of diabetes mellitus (DM). Using segmental (5 mm) femoral defects created in non-DM and DM rats and treated with allografts containing demineralized bone matrix (DBM) or DBM with MSC augmentation, we demonstrated that the presence of DM resulted in less mature bone 4 weeks after treating the defect with Allo DBM (P< 0.001). However, there was significantly more mature bone in the DM/MSC group when compared to the DM/DBM group at both 4 and 8 weeks (p<0.001 and p=0.004). Furthermore, significantly more bone formation was observed in the DM/MSC group compared to the DM/DBM group at the 4-week time point (p<0.001). The results of this study suggest that MSC are a potential adjunct for bone regeneration when implanted in an orthotopic site in the presence of DM. The primary strength of this study is that, to our knowledge, this is the first study exploring MSC as a potential adjuvant for allograft incorporation in a DM population. This study demonstrates the potential role of MSC as an adjunct, to address the clinical problems of delayed allograft incorporation in the presence of DM.

Summary of the Significance of the Work

Critically sized bone defects often occur as a result of metabolic diseases, trauma, or cancer. Bone lesions above a critical size become atrophic and scarred rather than regenerate, leading to nonunion, and may require revision surgery to restore lost function. More than one million orthopedic operations are performed in the United States each year for reconstructive surgery,

trauma, or abnormal skeletal defects. Often, large amounts of autologous, or alternatively, large bulk allograft (Allo) are needed in the surgical procedure to achieve the reconstructive goals. Although the current technique for bone grafting is autologous cancellous bone, various problems are associated with its acquisition, including donor site morbidity, loss of function, and a limited supply. Another major issue affecting both autograft and Allo incorporation is the presence of systemic diseases like diabetes mellitus (DM). Currently, 21 million Americans are diagnosed with DM, with over 600,000 new cases diagnosed each year. Despite the paucity of literature on the impact of DM on bone formation and fracture healing, the few clinical series demonstrate a significant deleterious effect of this systemic disease. DM patients experience a significant delay to union, uniting at an average of approximately two times longer than fractures in patients without DM. Our research is especially valuable because we demonstrated that rhBMP-2 can be utilized to significantly enhance bone healing response in diabetic segmental defect model. We also demonstrated that Mesenchymal Stem Cells (MSC) augmentation accelerate bone allograft incorporation in the presence of diabetes mellitus. Overall, our study promotes the use of rhBMP-2 and MSC augmentation in bone regeneration and formation and strongly support continued research in the use of osteogenic markers, non-osseous carriers for MSC to decrease the amount of bone needed for reconstructive surgery. Additional studies may focus on application of temporal control of genetic protein release for the determination of which specific phase of bone remodeling is up-regulated.

Summary of the Implementation/Influence of the Work

My 3 papers related to this project have been widely cited, with a total of 99 citations. For specific citation details, please refer to my google scholar profile. A few notable citations include:

Yu, W. Zhou, Y. Song et al. 2011. “Development of mesenchymal stem cell-implant complexes by cultured cells sheet enhances osseointegration in type 2 diabetic rat model”. Bone. 2011 September; 49(3): 387-394. – In this study, the researchers developed a mesenchymal stem cells (MSC)-implant complex as a novel tissue engineering approach that promotes osseous healing in the presence of diabetes mellitus type 2. These researchers from the Fourth Military Medical University (Xi’an, PR China) cited one of my papers and specifically mentioned our study in the context of demonstrating that their findings were similar to ours and support that MSCs may accelerate bone healing in the DM rat model.
K. Udehiya, Amarpal, H.P. Aithal et al. 2013. “Comparison of autogenic and allogenic bone marrow derived mesenchymal stem cells for repair of segmental bone defects in rabbits”. Research in Veterinary Science. June 2013; 94(3): 743-752. – In this study, the researchers compared the use of Autogenic and Allogenic bone marrowderived mesenchymal stem cells (BM-MSCs) for repair of bone gap defect in rabbits. These researchers from the Indian Veterinary Research Institute (Izatnagar, India) cited one of my papers and specifically mentioned our study in the context of demonstrating that their findings were similar to ours, specifically observing more bony union in mesenchymal stem cells treated group as compared to the groups treated by demineralized bone matrix (DBM) in diabetic animals at the 4 week time point, and that the newly formed bone showed early initiation of remodeling in treatment groups.
I. Ko, L.S. Coimbra, C. Tian et al. 2015. “Diabetes reduces mesenchymal stem cells in fracture healing through a TNFα-mediated mechanism”. Diabetologia. March 2015; 58(3): 633-642. – This work was supported by National Institute of Health/National Institute of Arthritis and Muscular and Skin Diseases (NIH/NIAMS) grant. Inthis study, the researchers demonstrated that Diabetes significantly increased TNFα levels and reduced MSC numbers in new bone area. The researchers suggested that reducing the activity of TNFα in vivo may help to preserve endogenous MSCs and maximize regenerative potential in diabetic patients. These researchers from University of Pennsylvania (Philadelphia, USA) cited one of my papers and specifically mentioned our study in the context of demonstrating that the treatment of diabetic fractures with MSCs can promote healing.
M. Wojtowicz, A. Shekaran, M.E. Oest et al. 2010. “Coating of biomaterial scaffolds with the collagen-mimetic peptide GFOGER for bone defect repair”. Biomaterials.March 2010; 31(9): 2574-2582. – This work was supported by the National Institutes of Health and Georgia Tech/Emory National Science Foundation ERC on the Engineering of Living Tissues. Inthis study, the collagen-mimetic peptide, GFOGER, was used by the researchers to coat synthetic PCL scaffolds to promote bone formation in critically-sized segmental defects in rats. The researchers demonstrated a simple, cell/growth factor-free strategy to promote bone formation in challenging, non-healing bone defects. These researchers from Georgia Institute of Technology (Atlanta, GA, USA) cited one of my papers and specifically mentioned our study in the context of demonstrating that although the GFOGER sequence is present in native full-length collagen, collagen alone does not enhance bone formation in critically-sized defects.
Shekaran, J. Garcia, A. Clark et al. 2014. “Bone regeneration using an alpha 2 beta 1 integrin-specific hydrogel as BMP-2 delivery vehicle”. Biomaterials.July 2014; 35(21): 5453-5461. – This work was funded by the National Institutes of Health, USA and by the Singaporean Agency for Science, Technology and Research. In this study, the researchers engineered a protease-degradable poly(ethylene glycol) (PEG) synthetic hydrogel functionalized with a triple helical, α2β1 integrin-specific peptide (GFOGER) as a BMP-2 delivery vehicle. The findings demonstrate that GFOGER hydrogels promote bone regeneration in challenging defects with low delivered BMP-2 doses and represent an effective delivery vehicle for protein therapeutics with translational potential. These researchers from Georgia Institute of Technology (Atlanta, GA, USA) cited one of my papers and specifically mentioned our study in the context of demonstrating the differences of healing induced by the GFOGER-tethered PEG gels compared to collagen foams.
S.R. Angle, K. Sena, D.R. Sumner et al. 2012. “Healing of rat femoral segmental defect with bone morphogenetic protein-2: A dose response study”. Journal of Musculoskelet Neuronal Interact. 2012; 12(1): 28-37. – This work was funded in parts by the National Institutes of Health – National Institute of Arthritis and Musculoskeletal and Skin Diseases, and a Medtronic grant from the Orthopaedic Trauma Association. In this study, the researchers used a segmental defect model to assess the dose response of rhBMP-2 using quantitative and qualitative endpoints. The researchers demonstrated that all doses of rhBMP-2 result in new bone formation. However, there is an optimum dose of 12 μg of rhBMP-2 for bone repair in the model used, above which and below which less stimulation of bone occurs. These researchers from Rush University Medical Center (Chicago, IL, USA) and University of Illinois (Chicago, IL, USA) cited one of my papers and specifically mentioned our study in the context of demonstrating that Recombinant human BMP-2 (rhBMP-2) has been most successful for bone repair in animal models.