INTRODUCTION: Posterolateral lumbar arthrodesis is the most common type of implemented fusion; however, the failure rate can be as high as 40%. Autologous bone grafting remains the gold standard material to promote bone formation despite some disadvantages. Bone allograft is obtained from human cadaveric specimens that undergoes rigorous preparation (i.e., decellularization and/or demineralization). The processed allograft lacks osteogenic cells and has limited osteoinductive properties. Some adjuvant therapies include supplementing the allograft with growth factors and/or adding stem cells. The purposes of this study are: (1) Determine whether in vitro human skin fibroblasts derived induced pluripotent stem cells-mesenchymal progenitors (iPSC-MPs) can adhere, remain viable, and undergo osteogenic differentiation on human bone allografts; and (2), whether iPSC-MPs seeded versus unseeded human bone graft implantation between two vertebrae promote and enhance in vivo spine fusion in a rat model.
METHODS: Human induced pluripotent stem cells (iPSCs) were generated from skin fibroblasts of a 50-year-old female (American Type Culture Collection; ATCC), using a mRNA-based reprogramming approach. iPSCs were cultured in induction medium (KO-DMEM + 20% FBS, 2mM GlutaMAX, 0.1 mM nonessential amino acids, 0.1 mM ß-mercaptoethanol and 100U/ml P/S) for 1 week and passaged until they became homogenous for a fibroblast-like morphology. Cells were referred to as iPSC-MPs. iPSC-MPs characterization was performed and compared to bone marrow derived mesenchymal stem cells (BM-MSCs) by flow cytometry for CD90, CD73 and CD105 markers; osteogeneic potential by monolayer culture in complete culture medium (CCM) and osteogenic differentiation medium (ODM) for 28 days, followed by Alizarin-Red staining for calcium deposition. Cancellous human bone allografts sized 4.8 mm x 4.8 mm and weighed ~400mg were obtained from a 51-year old female cadaveric humeral head (Lonetree Medical Donation, LLC, CO). These were decellularized, freeze-dried and sterilized. Followed seeding with 1.2 x 106 iPSC-MPs (passage 8)/40µl of media and stimulation in CCM or ODM for 1 hour, to be evaluated in vitro and implanted in vivo. For the in vitro section, seeded bone allografts were cultured in CCM or ODM for 28 days. Evaluation consisted of LIVE/DEAD assay at 3hrs., 3 days, and 28 days; while osteogenesis was evaluated with alkaline phosphatase (ALP) activity, Hematoxylin-Eosin (H&E) and osteocalcin IHC staining. For the in vivo section, 12 twelve-weeks old, male, Nude Outbred Immunodeficient rats undergoing spine fusion surgeries at L4-5 with human bone graft implantation were divided in three treatment groups: A- iPSC-MPs in CCM (n=4), B- iPSC-MPs in ODM (n=4), and C- unseeded in ODM (n=4). At 12 weeks, euthanasia was performed, and the surgical site was explanted for Micro-Computed Tomography (Mirco-CT) and histological evaluation: H&E, Alcian Blue-Hematoxylin (ABH), IHC for osteocalcin, and IF for Human-Nuclei. All animal procedures were performed in accordance with an approved animal use protocol from the University of Colorado Institutional Animal Care and Use Committee. Explants were subject to Micro-CT. Volumetric measurements on the axial sections between the posterior region of the L5 vertebrae and the anterior region of the L4 vertebrae were performed and presented as a bone volume to total tissue volume ratio (BV/TV). Statistical analysis was performed with SigmaPlot v.11.0 (Systat Software Inc.). Intergroup comparison was performed with a one-way Analysis of Variance (ANOVA) test and Tukey’s post hoc analysis correction was used to adjust for multiple group comparisons. Results were expressed as mean ± SD, and a P-value of ≤0.05 was considered statistically significant.
RESULTS: Similarly, to BM-MSCs, flow cytometric analysis of cultured iPSC-MPs at 28 days (passage 5), indicated that cells expressed greater than 95% expression of CD90 and CD73. Expression of CD105, was 22.6 ± 8.48% for iPSC-MPs and 93.3 ± 3.7% for BM-MSCs (P≤0.05). At 28 days, culture expanded iPSC-MPs in ODM were positively stained with Alizarin-Red S, while failed to differentiate in CCM. In vitro, iPSC-MPs were adhered and remained viable on CCM and ODM cultured bone allografts as early as 3 hours after seeding and at 28 days (Figure 1). ALP activity was evident on iPSC-MP-seeded allografts, particularly in ODM (P≤0.05 vs unseeded). H&E and osteocalcin staining showed bone allografts seeded with iPSC-MPs in ODM to have greater tissue formation. In vivo, at time of euthanasia, lumbar spines in all three groups showed bone graft in place. Hand manipulation for clinical assessment of spine fusion was difficult to assess and inconclusive (Figure 2). Macroscopically, there was no synovial reaction or inflammation. Micro-CT showed new bone formation in all groups, but no intergroup difference for BV/TV: Group A, BV/TV 0.12 ± 0.03; Group B, BV/TV 0.12 ± 0.03; and Group C, BV/TV 0.12 ± 0.02 (P≥0.05). H&E and ABH staining showed signs of ongoing intramembranous (IO) and endochondral ossification (EO) in all three groups. However, the seeded groups were marginally superior, specially the ODM seeded (Group B) (Figure 3). IHC for osteocalcin stained positive and was more notorious in groups A and B, evidencing osteoid deposition and IO. Moreover, groups A and B were more cellular than group C. Despite presenting more cellularity, IF for Human-Nuclei, showed only isolated positive cells in very few sections for groups A and B, but not for C. In these cases, the positive cells were found deep in the graft, reflecting scarce survival of human iPSC-MPs at 12 weeks.
DISCUSSION: This pilot study proved that human iPSCs can be directed towards mesenchymal and osteogeneic lineage in monolayer culture. They can attach and remain viable on bone allografts, indicating revitalization. In vivo, at 12 weeks, the presence of human bone graft seeded with iPSC-MPs in CCM or ODM have shown slightly superior osteogeneic capacity to unseeded bone graft, as reflected by ongoing IO and EO. Intriguingly, the Micro-CT showed no intergroup difference despite histological differences. Scarce human iPSC-MPs survived, but, whether these differentiated or not towards osteoblasts and osteocytes, remained unknown. However, histologically seeded grafts had more cellular connective tissue with trabecular bone in different stages of maturity, hence, most bone formation was of host origin. This suggests that iPSC-MPs secrete paracrine factors that enhance tissue regeneration and osteogenesis from migrating progenitor and stem cells found in the host’s neighboring receptive tissue and/or bone marrow. Limitations include a small sample size, a one-time point evaluation, no iPSC-MPs quantification and no testing in an immunocompetent group. However, this exceeded the aim of this study and we decided to run this study on immunosuppressive rats to avoid any potential confounding variables. Factors to be considered for future studies. In addition, spine fusion remained clinically inconclusive, but the bone allografts can become osteogeneic and promote bone formation when seeded with iPSC-MPs.
SIGNIFIFCANCE: This pilot study shows that human fibroblast derived iPSC-MPs are a novel candidate, safe and alternative biological tool for enhancing bone allografts bone formation capacity. They proved to adhere, remain viable and maintain pluripotency in vitro and in vivo. This study encourages further research in immunocompetent. These innovative cells could pave the way to create patient-specific bone constructs for orthopedic regenerative medicine.