Calcium Phosphate Cement Scaffolds Research Articles

Novel magnetic calcium phosphate-stem cell construct with magnetic field enhances osteogenic differentiation and bone tissue engineering

Superparamagnetic iron oxide nanoparticles (IONPs) are promising bioactive additives to fabricate magnetic scaffolds for bone tissue engineering. To date, there has been no report on osteoinductivity of IONP-incorporated calcium phosphate cement (IONP-CPC) scaffold on stem cells using an exterior static magnetic field (SMF). The objectives of this study were to: (1) develop a novel magnetic IONP-CPC construct for bone tissue engineering, and (2) investigate the effects of IONP-incorporation and SMF application on the proliferation, osteogenic differentiation and bone mineral synthesis of human dental pulp stem cells (hDPSCs) seeded on IONP-CPC scaffold for the first time. The novel magnetic IONP-CPC under SMF enhanced the cellular performance of hDPSCs, yielding greater alkaline phosphatase activities (about 3-fold), increased expressions of osteogenic marker genes, and more cell-synthesized bone minerals (about 2.5-fold), compared to CPC control and nonmagnetic IONP-CPC. In addition, IONP-CPC induced more active osteogenesis than CPC control in rat mandible defects. These results were consistent with the enhanced cellular performance by magnetic IONP in media under SMF. Moreover, nano-aggregates were detected inside the cells by transmission electron microscopy (TEM). Therefore, the enhanced cell performance was attributed to the physical forces generated by the magnetic field together with cell internalization of the released magnetic nanoparticles from IONP-CPC constructs.

Evaluation of Bone Sialoprotein Coating of Three-Dimensional Printed Calcium Phosphate Scaffolds in a Calvarial Defect Model in Mice.

The bioactive coating of calcium phosphate cement (CPC) is a promising approach to enhance the bone-healing properties of bone substitutes. The purpose of this study was to evaluate whether coating CPCs with bone sialoprotein (BSP) results in increased bone formation. Forty-five female C57BL/6NRj mice with an average age of six weeks were divided into three groups. Either a BSP-coated or an uncoated three-dimensional plotted scaffold was implanted into a drilled 2.7-mm diameter calvarial defect, or the defect was left empty (control group; no CPC). Histological analyses revealed that BSP-coated scaffolds were better integrated into the local bone stock eight weeks after implantation. Bone volume/total volume (BV/TV) ratios and bone thickness at the bone–implant contact were analyzed via micro computed tomography (µCT) after eight weeks. BSP-coated scaffolds and uncoated CPC scaffolds increased bone thickness in comparison to the control (CPC + BSP: 691.1 ± 253.5 µm, CPC: 603.1 ± 164.4 µm, no CPC: 261.7 ± 37.8 µm, p < 0.01). Accordingly, BV/TV was enhanced in both scaffold groups (CPC + BSP: 1.3 ± 0.5%, CPC: 0.9 ± 0.5%, no CPC: 0.2 ± 0.3%, p < 0.01). The BSP coating showed a tendency towards an increased bone thickness (p = 0.18) and BV/TV (p = 0.18) in comparison to uncoated CPC scaffolds. However, a significant increase in bone formation through BSP coating was not found.

A Methylcellulose Hydrogel as Support for 3D Plotting of Complex Shaped Calcium Phosphate Scaffolds.

3D plotting is an additive manufacturing technology enabling biofabrication, thus the integration of cells or biologically sensitive proteins or growth factors into the manufacturing process. However, most (bio-)inks developed for 3D plotting were not shown to be processed into clinically relevant geometries comprising critical overhangs and cavities, which would collapse without a sufficient support material. Herein, we have developed a support hydrogel ink based on methylcellulose (mc), which is able to act as support as long as the co-plotted main structure is not stable. Therefore, 6 w/v %, 8 w/v % and 10 w/v % mc were allowed to swell in water, resulting in viscous inks, which were characterized for their rheological and extrusion properties. The successful usage of 10 w/v % mc as support ink was proven by multichannel plotting of the support together with a plottable calcium phosphate cement (CPC) acting as main structure. CPC scaffolds displaying critical overhangs or a large central cavity could be plotted accurately with the newly developed mc support ink. The dissolution properties of mc allowed complete removal of the gel without residuals, once CPC setting was finished. Finally, we fabricated a scaphoid bone model by computed tomography data acquisition and co-extrusion of CPC and the mc support hydrogel.

Bioprinting of mineralized constructs utilizing multichannel plotting of a self-setting calcium phosphate cement and a cell-laden bioink

Due to their characteristic resemblance of the mineral component of bone, calcium phosphates are widely accepted as optimal bone substitute materials. Recent research focused on the development of pasty calcium phosphate cement (CPC) formulations, which can be fabricated into various shapes by low-temperature extrusion-based additive manufacturing, namely 3D plotting. While it could be demonstrated that sensitive substances like growth factors can be integrated in such printed CPC scaffolds without impairment of their biological activity live cells cannot be suspended in CPC as they may not be functional when enclosed in a solid and stiff matrix. In contrast, 3D bioprinting of soft cell-laden hydrogels (bioinks) enables the fabrication of constructs with spatially defined cell distribution, which has the potential to overcome problems of conventional cell seeding techniques—but such objects lack mechanical stability. Herein, we combine 3D plotting of CPC and bioprinting of a cell-laden bioink for the first time. As model bioink, an alginate-methylcellulose blend (alg/mc) was used, previously developed by us. Firstly, a fabrication regime was established, enabling optimal setting of CPC and cell survival inside the bioink. As the cells are exposed to the chemical changes of CPC precursors during setting, we studied the compatibility of the complex system of CPC and cell-laden alg/mc for a combined extrusion process and characterized the cellular behavior of encapsulated human mesenchymal stroma cells within the bioink at the interface and in direct vicinity to the CPC. Furthermore, biphasic scaffolds were mechanically characterized and a model for osteochondral tissue grafts is proposed. The manuscript discusses possible impacts of the CPC setting reaction on cells within the bioink and illustrates the advantages of CPC in bioprinting as alternative to the commonly used thermoplasts for bone tissue engineering.

Endosteal and Perivascular Subniches in a 3D Bone Marrow Model for Multiple Myeloma.

The bone marrow microenvironment is the preferred location of multiple myeloma, supporting tumor growth and development. It is composed of a collection of interacting subniches, including the endosteal and perivascular niche. Current in vitro models mimic either of these subniches. By developing a model combining both niches, this study aims to further enhance the ability to culture primary myeloma cells in vitro. Also, the dependency of myeloma cells on each niche was studied. A 3D bone marrow model containing two subniches was created using 3D bioprinting technology. We used a bioprintable pasty calcium phosphate cement (CPC) scaffold with seeded osteogenic multipotent mesenchymal stromal cells (O-MSCs) to model the endosteal niche, and Matrigel containing both endothelial progenitor cells (EPCs) and MSCs to model the perivascular niche. Within the model containing one or both of the niches, primary CD138+ myeloma cells were cultured and analyzed for both survival and proliferation. The 3D bone marrow model with combined subniches significantly increasing the proliferation of CD138+ myeloma cells compared to both environments separately. The developed model showed an essential role of the perivascular niche over the endosteal niche in supporting myeloma cells. The developed model can be used to study the expansion of primary myeloma cells and their interactions with varying bone marrow subniches.

Encapsulation of mesenchymal stem cells in chitosan/β-glycerophosphate hydrogel for seeding on a novel calcium phosphate cement scaffold

Due to its moldability, biocompatibility, osteoconductivity and resorbability, calcium phosphate cement (CPC) is a highly promising scaffold material for orthopedic applications. However, pH changes and ionic activity during the CPC setting reaction may adversely affect cells seeded directly on CPC. Moreover, a lack of macropores in CPC limits ingrowth of new bone. The objectives of this study were to prepare macroporous CPC scaffolds via porogen leaching, using mannitol crystals as the porogen and to evaluate the in vitro proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) encapsulated in chitosan/β-glycerophosphate (C/GP) hydrogel prior to exposure to the novel CPC scaffold. MSCs were found to be adhered to the surfaces of CPC macropores via scanning electron microscopy. The viability and osteogenic differentiation of MSCs in C/GP hydrogel with or without exposure to CPC constructs containing mannitol crystals indicated that coating with C/GP hydrogel protected the cells during cement mixing and setting. In conclusion, novel, macroporous CPC scaffolds were prepared, and our data indicate that a hydrogel encapsulation-based strategy can be used to protect cells during scaffold formation. Thus, the MSC-laden CPC scaffolds show promise for the delivery of stem cells to promote bone regeneration.

Localization and promotion of recombinant human bone morphogenetic protein-2 bioactivity on extracellular matrix mimetic chondroitin sulfate-functionalized calcium phosphate cement scaffolds

Localization of recombinant human bone morphogenetic protein-2 (rhBMP-2) with continuous and effective osteogenic stimulation is still a great challenge in the field of bone regeneration. To achieve this aim, rhBMP-2 was tethered on chondroitin sulfate (CS)-functionalized calcium phosphate cement (CPC) scaffolds through specific noncovalent interactions. CS, one of the core glycosaminoglycans, was covalently conjugated onto CPC scaffolds with the assistance of polydopamine (PDA) and further immobilized rhBMP-2 in a biomimetic form. The CPC-PDA-CS scaffolds not only controlled the release kinetics and presentation state of rhBMP-2 but also effectively increased the expression levels of bone morphogenetic protein receptors (BMPRs) and enhanced the recognitions of the remaining rhBMP-2 to BMPRs. Strikingly, the rhBMP-2-loaded CPC-PDA-CS significantly promoted the cellular surface translocation of BMPRs (especially BMPR-IA). In vivo studies demonstrated that, compared with the rhBMP-2 upon CPC and CPC-PDA, the rhBMP-2 upon CPC-PDA-CS exhibited sustained release and induced high quality and more ectopic bone formation. Collectively, these results suggest that rhBMP-2 can be localized within CS-functionalized CPC scaffolds and exert continuous, long-term, and effective osteogenic stimulation. Thus, this work could provide new avenues in mimicking bone extracellular matrix microenvironment and localizing growth factor activity for enhanced bone regeneration. Statement of SignificanceA bioinspired chondroitin sulfate (CS)-functionalized calcium phosphate cement (CPC) platform was developed to tether recombinant human bone morphogenetic protein-2 (rhBMP-2), which could exhibit continuous, long-term, and effective osteogenic stimulation in bone tissue engineering. Compared with rhBMP-2-loaded CPC, the rhBMP-2-loaded CPC-polydopamine-CS scaffolds induced higher expression of bone morphogenetic protein receptors (BMPRs), greater cellular surface translocation of bone morphogenetic protein receptor-IA, higher binding affinity of BMPRs/rhBMP-2, and thus higher activation of the drosophila gene mothers against decapentaplegic protein-1/5/8 (Smad1/5/8) and extracellular-regulated protein kinases-1/2 (ERK1/2) signaling. This work can provide new guidelines for the design of BMP-2-based bioactive materials for bone regeneration.

Injectable calcium phosphate scaffold with iron oxide nanoparticles to enhance osteogenesis via dental pulp stem cells

Literature search revealed no systematic report on iron oxide nanoparticle-incorporating calcium phosphate cement scaffolds (IONP-CPC). The objectives of this study were to: (1) use γFe2O3 nanoparticles (γIONPs) and αFe2O3 nanoparticles (αIONPs) to develop novel IONP-CPC scaffolds, and (2) investigate human dental pulp stem cells (hDPSCs) seeding on IONP-CPC for bone tissue engineering for the first time. IONP-CPC scaffolds were fabricated. Physiochemical properties of IONP-CPC scaffolds were characterized. hDPSC seeding on scaffolds, cell proliferation, osteogenic differentiation and bone matrix mineral synthesis by cells were measured. Our data demonstrated that the osteogenic differentiation of hDPSCs was markedly enhanced via IONP incorporation into CPC. Substantial increases (about three folds) in ALP activity and osteogenic gene expressions were achieved over those without IONPs. Bone matrix mineral synthesis by the cells was increased by two- to three folds over that without IONPs. The enhanced cellular osteogenesis was attributed to: (1) the surface nanotopography of IONP-CPC scaffold, and (2) the cell internalization of IONPs released from IONP-CPC scaffold. Our results demonstrate that the novel CPC functionalized with IONPs is promising to promote osteoinduction and bone regeneration. In conclusion, it is highly promising to incorporate γIONPs and αIONPs into CPC scaffold for bone tissue engineering, yielding substantially better stem cell attachment, spreading and osteogenic differentiation, and much greater bone mineral synthesis by the seeded cells. Therefore, novel CPC scaffolds containing γIONPs and αIONPs are promising for dental, craniofacial and orthopaedic applications to substantially enhance bone regeneration.

Effect of bone sialoprotein coated three-dimensional printed calcium phosphate scaffolds on primary human osteoblasts.

The combination of the two techniques of rapid prototyping 3D-plotting and bioactive surface functionalization is presented, with emphasis on the in vitro effect of Bone Sialoprotein (BSP) on primary human osteoblasts (hOBs). Our primary objective was to demonstrate the BSP influence on the expression of distinctive osteoblast markers in hOBs. Secondary objectives included examinations of the scaffolds' surface and the stability of BSP-coating as well as investigations of cell viability and proliferation. 3D-plotted calcium phosphate cement (CPC) scaffolds were coated with BSP via physisorption. hOBs were seeded on the coated scaffolds, followed by cell viability measurements, gene expression analysis and visualization. Physisorption is an effective method for BSP-coating. Coating with higher BSP concentrations leads to enhanced BSP release. Two BSP concentrations (50 and 200 μg/mL) were examined in this study. The lower BSP concentration (50 µg/mL) decreased ALP and SPARC expression, whereas the higher BSP concentration (200 μg/mL) did not change gene marker expression. Enhanced cell viability was observed on BSP-coated scaffolds on day 3. hOBs developed a polygonal shape and connected in an intercellular network under BSP influence. Quantitative cell morphology analyses demonstrated for BSP-coated CPCs an enhanced cell area and reduced circularity. The strength of the above-mentioned effects of BSP-coated scaffolds in vivo is unknown, and future work is focusing on bone ingrowth and vascularization in vivo. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2565-2575, 2018.

Calcium-Deficient Hydroxyapatite/Collagen/Platelet-Rich Plasma Scaffold with Controlled Release Function for Hard Tissue Regeneration.

Calcium phosphate cement scaffolds have been extensively used in bone tissue regeneration applications because they are similar to the inorganic components of bones and have outstanding biocompatibility. However, the main shortcomings of ceramic scaffolds are their brittleness and low retention level of various growth factors or drugs. To overcome these shortcomings, we have reinforced ceramic scaffolds using synthetic and natural polymers as composites. Here, we designed a new bone-biomimetic composite consisting of calcium deficient hydroxyapatite (CDHA), collagen, and plate-rich plasma (PRP) using low temperature printing process. In the biocomposite, we manipulated the release of the bioactive component, PRP, which has several growth factors, for a long culture period (up to 35 days) by using a polyphenol, tannic acid. The mesh-structured biocomposite showed structural stability. We also tested the in vitro biocompatibilities of the biocomposite using preosteoblasts (MC3T3-E1) and found that the biocomposite showed better cell growth and differentiation compared to the control (fabricated without PRP). Although effects of platelet-rich plasma have remained controversial, our results indicated controlled release of PRP with a biocomposite showed enhanced proliferation and differentiation of preosteoblast cells.

The Effects of Platelet-Derived Growth Factor-BB on Human Dental Pulp Stem Cells Mediated Dentin-Pulp Complex Regeneration.

Dentin‐pulp complex regeneration is a promising alternative treatment for the irreversible pulpitis caused by tooth trauma or dental caries. This process mainly relies on the recruitment of endogenous or the transplanted dental pulp stem cells (DPSCs) to guide dentin‐pulp tissue formation. Platelet‐derived growth factor (PDGF), a well‐known potent mitogenic, angiogenic, and chemoattractive agent, has been widely used in tissue regeneration. However, the mechanisms underlying the therapeutic effects of PDGF on dentin‐pulp complex regeneration are still unclear. In this study, we tested the effect of PDGF‐BB on dentin‐pulp tissue regeneration by establishing PDGF‐BB gene‐modified human dental pulp stem cells (hDPSCs) using a lentivirus. Our results showed that PDGF‐BB can significantly enhance hDPSC proliferation and odontoblastic differentiation. Furthermore, PDGF‐BB and vascular endothelial growth factor (VEGF) secreted by hDPSCs enhanced angiogenesis. The chemoattractive effect of PDGF‐BB on hDPSCs was also confirmed using a Transwell chemotactic migration model. We further determined that PDGF‐BB facilitates hDPSCs migration via the activation of the phosphatidylinositol 3 kinase (PI3K)/Akt signaling pathway. In vivo, CM‐DiI‐labeled hDPSCs were injected subcutaneously into mice, and our results showed that more labeled cells were recruited to the sites implanted with calcium phosphate cement scaffolds containing PDGF‐BB gene‐modified hDPSCs. Finally, the tissue‐engineered complexes were implanted subcutaneously in mice for 12 weeks, the Lenti‐PDGF group generated more dentin‐like mineralized tissue which showed positive staining for the DSPP protein, similar to tooth dentin tissue, and was surrounded by highly vascularized dental pulp‐like connective tissue. Taken together, our data demonstrated that the PDGF‐BB possesses a powerful function in prompting stem cell‐based dentin‐pulp tissue regeneration. Stem Cells Translational Medicine 2017;6:2126–2134

Engineering human bone grafts with new macroporous calcium phosphate cement scaffolds.

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.

Metformin induces osteoblastic differentiation of human induced pluripotent stem cell-derived mesenchymal stem cells.

Metformin, a first-line antidiabetic drug used by millions of patients, has been shown to have potential osteogenic properties. The present study was performed to test the hypothesis that clinically relevant doses of metformin promote the osteogenic differentiation and mineralization of induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs). iPSC-MSCs were treated with metformin (10 μm) to assess cell viability, osteogenic differentiation, mineralization and activation of the LKB1/AMP-activated protein kinase (AMPK) signalling pathway, a surrogate marker of metformin action. To determine its potential application in MSC-based bone and periodontal tissue engineering, iPSC-MSCs were also treated with metformin when seeded on to calcium phosphate cement (CPC) scaffolds. Immunoblotting and cellular uptake assays showed that iPSC-MSCs express functional organic cation transporter-1 (OCT-1), a transmembrane protein that mediates the intracellular uptake of metformin. Although metformin treatment did not impair iPSC-MSC viability, it significantly stimulated alkaline phosphatase activity, enhanced mineralized nodule formation and increased expression of osteogenic markers, including Runt-related transcription factor 2 (RUNX2) and osterix. Inhibition of LKB1 activity, a common upstream AMPK kinase, markedly reversed metformin-induced AMPK activation, RUNX2 expression and nuclear localization. Moreover, metformin substantially increased mineralized nodule formation of iPSC-MSC seeded on CPC scaffolds. Collectively, functional OCT-expressing iPSC-MSCs responded to metformin by inducing an osteogenic effect in part mediated by the LKB1/AMPK pathway. Considering the widespread use of metformin in diabetics, this work may lead to novel tissue-engineering platforms where autogenous OCT-expressing iPSC-MSCs might be used to enhance bone and periodontal regeneration in diabetic patients prescribed with daily doses of metformin.

Evaluation of Osteogenesis and Angiogenesis of Icariin in Local Controlled Release and Systemic Delivery for Calvarial Defect in Ovariectomized Rats

Typically, bone regenerative medicine is applied to repair bone defects in patients with osteoporosis. Meanwhile, there is an urgent need to develop safe and cheap drugs that induce bone formation. Icariin, which is reported to promote the osteogenesis of stem cells in vitro, is the main active component of Herba Epimedii. However, whether icariin could repair bone defects caused by osteoporosis remains unknown. In this study, an osteoporosis model in rats was established by an ovariectomy first, and then, the osteogenic and angiogenic differentiation of bone mesenchymal stem cells (BMSCs) treated with icariin was evaluated. Furthermore, calcium phosphate cement (CPC) scaffolds loaded with icariin were constructed and then implanted into nude mice to determine the optimal construction. To evaluate its osteogenic and angiogenic ability in vivo, this construction was applied to calvarial defect of the ovariectomized (OVX) rats accompanied with an icariin gavage. This demonstrated that icariin could up-regulate the expression of osteogenic and angiogenic genes in BMSCs. Meanwhile, osteoclast formation was inhibited. Moreover, CPC could act as a suitable icariin delivery system for repairing bone defects by enhancing osteogenesis and angiogenesis, while the systemic administration of icariin has an antiosteoporotic effect that promotes bone defect repair.

Strontium(II) and mechanical loading additively augment bone formation in calcium phosphate scaffolds.

Calcium phosphate cements (CPCs) are widely used for bone-defect treatment. Current developments comprise the fabrication of porous scaffolds by three-dimensional plotting and doting using biologically active substances, such as strontium. Strontium is known to increase osteoblast activity and simultaneously to decrease osteoclast resorption. This study investigated the short- and long-term in vivo performances of strontium(II)-doted CPC (SrCPC) scaffolds compared to non-doted CPC scaffolds after implantation in unloaded or load-bearing trabecular bone defects in sheep. After 6 weeks, both CPC and SrCPC scaffolds exhibited good biocompatibility and osseointegration. Fluorochrome labeling revealed that both scaffolds were penetrated by newly formed bone already after 4 weeks. Neither strontium doting nor mechanical loading significantly influenced early bone formation. In contrast, after 6 months, bone formation was significantly enhanced in SrCPC compared to CPC scaffolds. Energy dispersive X-ray analysis demonstrated the release of strontium from the SrCPC into the bone. Strontium addition did not significantly influence material resorption or osteoclast formation. Mechanical loading significantly stimulated bone formation in both CPC and SrCPC scaffolds after 6 months without impairing scaffold integrity. The most bone was found in SrCPC scaffolds under load-bearing conditions. Concluding, these results demonstrate that strontium doting and mechanical loading additively stimulated bone formation in CPC scaffolds and that the scaffolds exhibited mechanical stability under moderate load, implying good clinical suitability. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:106-117, 2018.

Angiogenic and osteogenic regeneration in rats via calcium phosphate scaffold and endothelial cell co-culture with human bone marrow mesenchymal stem cells (MSCs), human umbilical cord MSCs, human induced pluripotent stem cell-derived MSCs and human embryonic stem cell-derived MSCs.

Angiogenesis is a limiting factor in regenerating large bone defects. The objective of this study was to investigate angiogenic and osteogenic effects of co-culture on calcium phosphate cement (CPC) scaffold using human umbilical vein endothelial cells (hUVECs) and mesenchymal stem cells (MSCs) from different origins for the first time. hUVECs were co-cultured with four types of cell: human umbilical cord MSCs (hUCMSCs), human bone marrow MSCs (hBMSCs) and MSCs from induced pluripotent stem cells (hiPSC-MSCs) and embryonic stem cells (hESC-MSCs). Constructs were implanted in 8mm cranial defects of rats for 12weeks. CPC without cells served as control 1. CPC with hBMSCs served as control 2. Microcapillary-like structures were successfully formed on CPC in vitro in all four co-cultured groups. Microcapillary lengths increased with time (p<0.05). Osteogenic and angiogenic gene expressions were highly elevated and mineralization by co-cultured cells increased with time (p<0.05). New bone amount and blood vessel density of co-cultured groups were much greater than controls (p<0.05) in an animal study. hUVECs co-cultured with hUCMSCs, hiPSC-MSCs and hESC-MSCs achieved new bone and vessel density similar to hUVECs co-cultured with hBMSCs (p>0.1). Therefore, hUCMSCs, hiPSC-MSCs and hESC-MSCs could serve as alternative cell sources to hBMSCs, which require an invasive procedure to harvest. In conclusion, this study showed for the first time that co-cultures of hUVECs with hUCMSCs, hiPSC-MSCs, hESC-MSCs and hBMSCs delivered via CPC scaffold achieved excellent osteogenic and angiogenic capabilities in vivo. The novel co-culture constructs are promising for bone reconstruction with improved angiogenesis for craniofacial/orthopaedic applications. Copyright © 2017 John Wiley & Sons, Ltd.

Bone regeneration in minipigs via calcium phosphate cement scaffold delivering autologous bone marrow mesenchymal stem cells and platelet-rich plasma.

Macroporous calcium phosphate cement (CPC) with stem cell seeding is promising for bone regeneration. The objective of this study was to investigate the effects of co-delivering autologous bone marrow mesenchymal stem cells (BMSCs) and autologous platelet-rich plasma (PRP) in CPC scaffold for bone regeneration in minipigs for the first time. Twelve female adult Tibet minipigs (12-18months old) were used. A cylindrical defect with 10mm height and 8mm diameter was prepared at the femoral condyle. Two bone defects were created in each minipig, one at each side of the femoral condyle. Three constructs were tested: (1) CPC scaffold (CPC control); (2) CPC seeded with BMSCs (CPC-BMSC); (3) CPC seeded with BMSCs and PRP (CPC-BMSC-PRP). Two time points were tested: 6 and 12weeks (n=4). Good integration of implant with surrounding tissues was observed in all groups. At 12weeks, the CPC-BMSC-PRP group had significantly less residual CPC remaining in the defect than the CPC-BMSC group and the CPC control (p<0.05). The residual CPC volume for the CPC-BMSC-PRP group was half that of the CPC control. New bone formation for CPC-BMSC-PRP was more than two-fold that of the CPC control (p<0.05). CPC-BMSC-PRP had new blood vessel density that was nearly two-fold that of the CPC control (p<0.05). In conclusion, CPC scaffold with autologous BMSC-PRP doubled the new bone regeneration and blood vessel density in minipigs compared with the CPC control. In the present study, the new macroporous CPC system with co-delivered BMSC-PRP has been shown to promote scaffold resorption and bone regeneration in large defects. Copyright © 2017 John Wiley & Sons, Ltd.

Novel hiPSC-based tri-culture for pre-vascularization of calcium phosphate scaffold to enhance bone and vessel formation

Vascularization of tissue-engineered bone is a critical step in maintaining cell viability and advancing cell performance in vivo. In this study, a novel tri-culture system was developed to elicit pre-vascularization of calcium phosphate cement (CPC) scaffold in which human induced pluripotent stem cell-derived mesenchymal stem cells (hiPSMSCs) were seeded together with human umbilical vein endothelial cells (HUVECs) and pericytes. In a two-step methodology design, we first performed osteoinduction of the seeded hiPSMSCs on the CPC scaffold and then incorporated HUVECs and pericytes to the hiPSMSC-colonized CPC scaffold under a favorable culturing condition, with an objective to form a stable and functional capillary-like vascular network that sustained the engineered osseous tissue. The angiogenic and osteogenic effects of various culture strategies were studied and compared in nude rat model with cranial bone defects: (1) CPC scaffold alone (CPC control); (2) Pericyte-seeded CPC (CPC-pericytes); (3) HUVEC-seeded CPC (CPC-HUVECs); (4) hiPSMSC-seeded CPC (CPC-hiPSMSCs); (5) HUVECs co-cultured with hiPSMSCs on CPC (bi-culture group) and (6) HUVECs and pericytes co-cultured with hiPSMSCs on CPC (tri-culture group). After 12weeks, tri-culture group showed the highest amount of new bone (new bone area fraction of (45.3±2.7) %, p<0.05) and vessel formation (new blood vessel density of (50.7±3.8) vessels/mm2, p<0.05) in all groups. Our results demonstrated that the tri-culture strategy was effective in promoting angiogenesis and osteogenesis for bone tissue engineering.

Engineering bone regeneration with novel cell-laden hydrogel microfiber-injectable calcium phosphate scaffold

Cell-based tissue engineering is promising to create living functional tissues for bone regeneration. The implanted cells should be evenly distributed in the scaffold, be fast-released to the defect and maintain high viability in order to actively participate in the regenerative process. Herein, we report an injectable calcium phosphate cement (CPC) scaffold containing cell-encapsulating hydrogel microfibers with desirable degradability that could deliver cells in a timely manner and maintain cell viability. Microfibers were synthesized using partially-oxidized alginate with various concentrations (0–0.8%) of fibrinogen to optimize the degradation rate of the alginate-fibrin microfibers (Alg-Fb MF). A fibrin concentration of 0.4% in Alg-Fb MF resulted in the greatest enhancement of cell migration, release and proliferation. Interestingly, a significant amount of cell–cell contact along the long-axis of the microfibers was established in Alg-0.4%Fb MF as early as day 2. The injectable tissue engineered construct for bone reconstruct was fabricated by mixing the fast-degradable Alg-0.4%Fb MF with CPC paste at 1:1 volume ratio. In vitro study showed that cells re-collected from the construct maintained good viability and osteogenic potentials. In vivo study demonstrated that the hBMSC-encapsulated CPC-MF tissue engineered construct displayed a robust capacity for bone regeneration. At 12weeks after implantation, osseous bridge in the rat mandibular defect was observed in CPC-MF-hBMSCs group with a new bone area fraction of (42.1±7.8) % in the defects, which was >3-fold that of the control group. The novel tissue-engineered construct presents an excellent prospect for a wide range of dental, craniofacial and orthopedic applications.

Co-Seeding Human Endothelial Cells with Human-Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells on Calcium Phosphate Scaffold Enhances Osteogenesis and Vascularization in Rats.

A major challenge in repairing large bone defects with tissue-engineered constructs is the poor vascularization in the defect. The lack of vascular networks leads to insufficient oxygen and nutrients supply, which compromises the survival of seeded cells. To achieve favorable regenerative effects, prevascularization of tissue-engineered constructs by co-culturing of endothelial cells and bone cells is a promising strategy. The aim of this study was to investigate the effects of human-induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs) co-cultured with human umbilical vein endothelial cells (HUVECs) for prevascularization of calcium phosphate cement (CPC) scaffold on bone regeneration in vivo for the first time. HUVECs co-cultured with hiPSC-MSCs formed microcapillary-like structures in vitro. HUVECs promoted mineralization of hiPSC-MSCs on CPC scaffolds. Four groups were tested in a cranial bone defect model in nude rats: (1) CPC scaffold alone (CPC control); (2) HUVEC-seeded CPC (CPC-HUVEC); (3) hiPSC-MSC-seeded CPC (CPC-hiPSC-MSC); and (4) HUVECs co-cultured with hiPSC-MSCs on CPC scaffolds (co-culture group). After 12 weeks, the co-culture group achieved the greatest new bone area percentage of 46.38% ± 3.8% among all groups (p < 0.05), which was more than four folds of the 10.61% ± 1.43% of CPC control. In conclusion, HUVECs co-cultured with hiPSC-MSCs substantially promoted bone regeneration. The novel construct of HUVECs co-cultured with hiPSC-MSCs delivered via CPC scaffolds is promising to enhance bone and vascular regeneration in orthopedic applications.