Engineering human bone grafts with new macroporous calcium phosphate cement scaffolds

Abstract

Bone engineering opens the possibility to grow large amounts of tissue products by combining patient-specific cells with compliant biomaterials. Decellularized tissue matrices represent suitable biomaterials, but availability, long processing time, excessive cost, and concerns on pathogen transmission have led to the development of biomimetic synthetic alternatives. We recently fabricated calcium phosphate cement (CPC) scaffolds with variable macroporosity using a facile synthesis method with minimal manufacturing steps and demonstrated long-term biocompatibility in vitro. However, there is no knowledge on the potential use of these scaffolds for bone engineering and whether the porosity of the scaffolds affects osteogenic differentiation and tissue formation in vitro. In this study, we explored the bone engineering potential of CPC scaffolds with two different macroporosities using human mesenchymal progenitors derived from induced pluripotent stem cells (iPSC-MP) or isolated from bone marrow (BMSC). Biomimetic decellularized bone scaffolds were used as reference material in all experiments. The results demonstrate that, irrespective of their macroporosity, the CPC scaffolds tested in this study support attachment, viability, and growth of iPSC-MP and BMSC cells similarly to decellularized bone. Importantly, the tested materials sustained differentiation of the cells as evidenced by increased expression of osteogenic markers and formation of a mineralized tissue. In conclusion, the results of this study suggest that the CPC scaffolds fabricated using our method are suitable to engineer bone grafts from different cell sources and could lead to the development of safe and more affordable tissue grafts for reconstructive dentistry and orthopaedics and in vitro models for basic and applied research.

Segmental Additive Tissue Engineering

“…In this study, we have demonstrated that a similar approach can be applied toward the effective and reproducible construction of segmental bone grafts. Previous studies in our laboratory have shown that iPSC-MPs can be expanded to trillion of cells in just a few weeks time period 10 , 14 , thus allowing the engineering of segmental bone grafts for autologous reconstruction at a reasonable cost and in a relatively short period of time.…”

GMP-compatible and xeno-free cultivation of mesenchymal progenitors derived from human-induced pluripotent stem cells

“…This implies that large numbers of cells would need to be administered to achieve therapeutic efficacy. The same is true if MSCs are to be used to engineer tissue grafts for replacement therapies [17, 30, 31]. Unfortunately, human MSCs derived from adult tissues exhibit limited proliferation potential, and therapeutically, functional cells may not be available in sufficient numbers for every patient [3, 7, 11, 37, 40].…”

Hypothermic and cryogenic preservation of tissue‐engineered human bone

“…Human bone grafts ( n = 18) were engineered as previously described . Briefly, human iPSC‐derived mesenchymal progenitor cells (line 1013A) at passage 6 were plated onto gelatin‐coated plasticware and expanded in medium consisting of high‐glucose KnockOut Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 20% (v/v) HyClone™ fetal bovine serum (FBS; GE Healthcare Life Sciences), fibroblast growth factor basic (1 ng/mL; Invitrogen), nonessential amino acids (0.1 mM; Gibco), glutaMAX (2 mM; Gibco), beta‐mercaptoethanol (0.1 mM; Gibco), and antibiotic‐antimycotic (100 U/mL; Gibco).…”

“…Briefly, human iPSC‐derived mesenchymal progenitor cells (line 1013A) at passage 6 were plated onto gelatin‐coated plasticware and expanded in medium consisting of high‐glucose KnockOut Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 20% (v/v) HyClone™ fetal bovine serum (FBS; GE Healthcare Life Sciences), fibroblast growth factor basic (1 ng/mL; Invitrogen), nonessential amino acids (0.1 mM; Gibco), glutaMAX (2 mM; Gibco), beta‐mercaptoethanol (0.1 mM; Gibco), and antibiotic‐antimycotic (100 U/mL; Gibco). Following expansion, the cells were detached using 0.25% trypsin‐ethylenediaminetetraacetic acid (Gibco), counted using a hematocytometer, and seeded onto sterile decellularized cow bone disks (8 mm in diameter and 5 mm in height) at a density of 10 6 cells per scaffold using a drop technique . Following seeding, the samples were cultured in an osteogenic environment consisting of high‐glucose DMEM medium supplemented with 10% (v/v) HyClone FBS (GE Healthcare Life Sciences), dexamethasone (1 µM; Sigma), beta‐glycerophosphate (10 µM; Sigma), ascorbic acid‐2‐phosphate (50 µM; Sigma, A8960), and antibiotic‐antimycotic (100 U/mL; Gibco) for 5 weeks.…”

“…Human iPSCs can be derived for every patient in virtually unlimited numbers, and represent a single cell source with the ability to differentiate into all of the specialized cells constituting the bone tissue . We recently engineered bone grafts by culturing human iPSC‐derived mesenchymal progenitors onto biomimetic scaffolds in bioreactors . In the present study, we asked whether these grafts could be preserved to allow distribution and banking, and studied the effects of two preservation methods on the tissue quality.…”