In VivoEctopic Bone Formation by Devitalized Mineralized Stem Cell Carriers Produced Under Mineralizing Culture Condition

Abstract

Functionalization of tissue engineering scaffolds with in vitro–generated bone-like extracellular matrix (ECM) represents an effective biomimetic approach to promote osteogenic differentiation of stem cells in vitro. However, the bone-forming capacity of these constructs (seeded with or without cells) is so far not apparent. In this study, we aimed at developing a mineralizing culture condition to biofunctionalize three-dimensional (3D) porous scaffolds with highly mineralized ECM in order to produce devitalized, osteoinductive mineralized carriers for human periosteal-derived progenitors (hPDCs). For this, three medium formulations [i.e., growth medium only (BM1), with ascorbic acid (BM2), and with ascorbic acid and dexamethasone (BM3)] supplemented with calcium (Ca2+) and phosphate (PO43−) ions simultaneously as mineralizing source were investigated. The results showed that, besides the significant impacts on enhancing cell proliferation (the highest in BM3 condition), the formulated mineralizing media differentially regulated the osteochondro-related gene markers in a medium-dependent manner (e.g., significant upregulation of BMP2, bone sialoprotein, osteocalcin, and Wnt5a in BM2 condition). This has resulted in distinguished cell populations that were identifiable by specific gene signatures as demonstrated by the principle component analysis. Through devitalization, mineralized carriers with apatite crystal structures unique to each medium condition (by X-ray diffraction and SEM analysis) were obtained. Quantitatively, BM3 condition produced carriers with the highest mineral and collagen contents as well as human-specific VEGF proteins, followed by BM2 and BM1 conditions. Encouragingly, all mineralized carriers (after reseeded with hPDCs) induced bone formation after 8 weeks of subcutaneous implantation in nude mice models, with BM2-carriers inducing the highest bone volume, and the lowest in the BM3 condition (as quantitated by nano-computed tomography [nano-CT]). Histological analysis revealed different bone formation patterns, either bone ossicles containing bone marrow surrounding the scaffold struts (in BM2) or bone apposition directly on the struts' surface (in BM1 and BM3). In conclusion, we have presented experimental data on the feasibility to produce devitalized osteoinductive mineralized carriers by functionalizing 3D porous scaffolds with an in vitro cell-made mineralized matrix under the mineralizing culture conditions.

Embryonic‐Like Mineralized Extracellular Matrix/Stem Cell Microspheroids as a Bone Graft Substitute

“…Higher expression of BMP‐2 was detected in the MECS group compared with the CS group, and osteogenic induction significantly increased BMP‐2 expression levels in both groups. These findings indicate that mineralized ECM could provide a cell‐instructive structural framework that induces stem cell osteogenic differentiation …”

Harnessing the Osteogenicity of In Vitro Stem Cell-Derived Mineralized Extracellular Matrix as 3D Biotemplate to Guide Bone Regeneration

“…33,34 In fact, our previous study has provided a ''proof-of-principle'' that 3D porous scaffolds bearing cellsecreted mineralized ECM were readily recognized by the host environment as an ''osteogenic biotemplate'' and triggered bone formation. 21 The limited bone formation on the carriers (<1% per total scaffold void volume of 64.8 mm 3 ) served as our motivation to further optimize the mineralizing culture condition in this study. As a result, we showed that the current modified culture protocol (i.e., combining the priming followed by a direct mineralization phase) augmented nearly five-fold ectopic bone formation on the carriers as compared with the previously reported direct culture mineralization method.…”

“…In this study, we assessed the ectopic bone induction and critical-sized calvarial defect bridging capacities of the produced devitalized mineralized carriers by using an immunocompromised large rodent model, that is, athymic nude rats (as compared with only ectopic implantation in nude mice in our previous study 21 ). This model was beneficial to avoid immunorejection of the hPDC-seeded carriers due to untoward immune responses on the transplanted human cells.…”

“…Color images available online at www.liebertpub.com/tea produced devitalized mineralized carriers with a higher ectopic bone-forming capacity (nearly five-folds higher) than that previously reported in our proof-of-principle study. 21…”

“…We recently reported the proof of principle of ectopic bone induction by these devitalized mineralized carriers (in combination with hPDCs). 21 This direct mineralizing culture protocol was based on simultaneous calcium (Ca) and phosphate (P) supplementation in culture medium to directly provoke active culture mineralization. However, the percentage of bone induced was far from trivial for clinical applications (<1% based on the calculated bone volume per total scaffold void volume of 64.8 mm 3 ), an observation that we believe was attributed to a low amount of deposited mineralized ECM due to direct induction of culture mineralization that limited further cell growth and ECM deposition.…”