Approximately 10% of fractures fail to heal without intervention, incurring substantial costs and necessitating current treatments which often present drawbacks. Bone morphogenetic protein-2 (BMP2) is effective but carries risks of life-threatening inflammation and ectopic bone formation. Autologous bone grafts, while the gold standard, suffer from limited availability and donor site morbidity. Synthetic and cadaveric bone products, though abundant and inexpensive, exhibit poor biocompatibility and batch-to-batch variation. The development of a safe, manufacturable bone mimic is a primary goal of bone tissue engineering, yet remains challenging. Human mesenchymal stem cells (hMSCs), due to their osteoinductive and immunomodulatory properties, are promising candidates for bone engineering. However, their clinical translation is hindered by donor variability and limited availability. Induced pluripotent stem cells (iPSCs) offer a theoretically limitless and reproducible source of cells, overcoming the limitations of BM-derived hMSCs. Previous research demonstrated that extracellular matrix (ECM) secreted by osteogenically enhanced hMSCs (OEhMSCs) improves bone healing. This study investigates the osteogenic potential of iPSC-derived hMSCs (ihMSCs) and the osteogenic matrix they produce (ihOCM), evaluating its efficacy in a murine calvarial defect model compared to BMP2.

The literature extensively documents the challenges associated with current bone fracture treatment modalities. The limitations of BMP2, autografts, and synthetic bone substitutes have been widely reported, highlighting the need for improved biomaterials. Studies on the use of mesenchymal stem cells (MSCs) in bone regeneration showcase their potential but also underscore the issues of donor variability and limited cell availability. The use of induced pluripotent stem cells (iPSCs) as a source of MSCs has been explored, with several studies demonstrating their osteogenic potential. However, this study aims to build upon these previous findings by focusing on a specific iPSC-derived MSC line and the unique properties of the extracellular matrix it produces.

This study employed a comprehensive methodology involving in vitro and in vivo experiments. Initially, the characteristics of ihMSCs derived from iPSCs were thoroughly characterized, assessing their proliferation rate, colony-forming capacity, immunophenotype, and trilineage differentiation potential (osteogenic, adipogenic, and chondrogenic) using techniques like flow cytometry, colony assays, and staining assays (Alizarin Red S, Oil Red O, Toluidine Blue). The role of the canonical Wnt (cWnt) pathway and PPARγ axis in ihMSC osteogenesis was investigated through subcellular fractionation, immunoblotting, and quantitative RT-PCR. Osteogenic enhancement was achieved using the PPARγ inhibitor GW9662. Extracellular matrix (ihOCM) was purified from OEihMSCs and its composition analyzed using proteomic analysis (LC-MS) and scanning electron microscopy (SEM) with energy-dispersive X-ray spectroscopy (EDS). To assess the role of collagens VI and XII, stable knockdown cell lines (KD6 and KD12) were generated using lentiviral shRNA constructs. In vivo experiments utilized a murine calvarial defect model. Immune-compromised nude mice received calvarial defects treated with either scrambled, KD6, or KD12 OEhMSCs, or with ihOCM in the presence or absence of cells, and compared to a BMP2 control. Bone healing was assessed using micro-computed tomography (µCT) and histological analysis (Masson's trichrome, H&E, TRAP). Radiomorphometric parameters (BMD and surface:volume ratio) were used to assess the quality of the newly formed bone. Statistical analyses included t-tests and ANOVA with appropriate post-hoc tests.

ihMSCs exhibited superior osteogenic properties compared to bone marrow-derived hMSCs, generating significantly larger quantities of osteogenic matrix (ihOCM) in vitro. Proteomic analysis revealed that ihOCM contained enriched levels of collagens VI and XII. Knockdown studies confirmed that both collagens VI and XII are crucial for osteogenesis, impacting proliferation and osteogenic potential of hMSCs both in vitro and in vivo. In the murine calvarial defect model, ihOCM demonstrated markedly superior bone healing capabilities compared to BMP2 at a standard effective dose. Remarkably, ihOCM alone, without the addition of cells, showed the highest healing index, surpassing all other treatment groups including BMP2 and cell-ihOCM combinations. Radiomorphometric analysis indicated that the newly formed bone in ihOCM-treated defects possessed comparable density and structural characteristics to native calvarial bone.

The findings demonstrate the remarkable osteoregenerative capacity of ihOCM derived from a single, reproducible iPSC line. The superior performance of ihOCM compared to BMP2 suggests a potential safer and more effective alternative for bone regeneration. The unexpected finding that ihOCM alone is more effective than ihOCM with added cells challenges the prevailing assumption that high cell numbers are essential for optimal bone healing. This might indicate that the ihOCM provides an optimal microenvironment that supports bone regeneration independent of the requirement for large numbers of exogenously added cells. The critical role of collagens VI and XII, as demonstrated by the knockdown studies, highlights their involvement in the osteogenic signaling within the ihOCM. Further research is needed to fully elucidate the mechanisms underlying the interaction between these collagens and other matrix components and their effect on osteogenesis.

This study successfully generated and characterized a potent osteoregenerative matrix (ihOCM) derived from a reproducible iPSC-derived MSC line. ihOCM exhibited superior bone healing capacity compared to BMP2 in a murine calvarial defect model, particularly when administered without additional cells. Collagens VI and XII were identified as key components mediating the osteogenic effects of ihOCM. This cell-free matrix offers a promising alternative to current bone regeneration therapies, warranting further investigation in larger animal models and eventually clinical trials. Future research should focus on elucidating the precise mechanisms of action of ihOCM and optimizing its manufacturing for clinical translation.

The study was conducted using a murine calvarial defect model, which may not fully replicate the complexities of human bone healing in different anatomical locations. The 4-week duration of the in vivo study may not capture the complete bone remodeling process. Further studies in larger animal models and long-bone defects are necessary to confirm the findings. While the ihOCM demonstrated excellent biocompatibility in this study, long-term biocompatibility and safety assessments are needed prior to clinical translation.