Calcium Phosphate Cement Scaffolds Research Articles

Hydrogel fibers encapsulating human stem cells in an injectable calcium phosphate scaffold for bone tissue engineering

Human induced pluripotent stem cells (hiPSCs), human embryonic stem cells (hESCs) and human umbilical cord mesenchymal stem cells (hUCMSCs) are exciting cell sources for use in regenerative medicine. There have been no reports on long hydrogel fibers encapsulating stem cells inside an injectable calcium phosphate cement (CPC) scaffold for bone tissue engineering. The objectives of this study were: (1) to develop a novel injectable CPC construct containing hydrogel fibers encapsulating cells for bone engineering, and (2) to investigate and compare cell viability, proliferation and osteogenic differentiation of hiPSC-MSCs, hESC-MSCs and hUCMSCs in injectable CPC. The pastes encapsulating the stem cells were fully injectable under a small injection force, and the injection did not harm the cells, compared with non-injected cells (p > 0.1). The mechanical properties of the stem cell–CPC construct were much better than those of previous injectable polymers and hydrogels for cell delivery. The hiPSC-MSCs, hESC-MSCs and hUCMSCs in hydrogel fibers in CPC had excellent proliferation and osteogenic differentiation. All three cell types yielded high alkaline phosphatase, runt-related transcription factor, collagen I and osteocalcin expression (mean ± SD; n = 6). Cell-synthesized minerals increased substantially with time (p < 0.05), with no significant difference among the three types of cells (p > 0.1). Mineralization by hiPSC-MSCs, hESC-MSCs and hUCMSCs in CPC at 14 d was 13-fold that at 1 d. In conclusion, all three types of cells (hiPSC-MSCs, hESC-MSCs and hUCMSCs) in a CPC scaffold showed high potential for bone tissue engineering, and the novel injectable CPC construct with cell-encapsulating hydrogel fibers is promising for enhancing bone regeneration in dental, craniofacial and orthopedic applications.

Bone morphogenetic protein 2 promotes osteogenesis of bone marrow stromal cells in type 2 diabetic rats via the Wnt signaling pathway

Type 2 diabetes mellitus impairs osteogenesis in bone marrow stromal cells (BMSCs). Bone morphogenetic protein 2 (BMP2) has been extensively applied for bone defect restoration and has been shown to activate the Wnt signaling pathway. The objective of this study was to investigate the effects of BMP2 on the cell proliferation and osteogenesis of type 2 diabetic BMSCs in rats and explore whether BMP2 induced osteogenesis via the stimulation of Wnt signaling pathway. The cell experiments were divided into DM (diabetic BMSCs), BMP25 (induced with 25ng/ml BMP2), BMP100 (induced with 100ng/ml BMP2) and BMP25 +XAV groups. All cells with or without the different concentrations of BMP2 were cultured under the same experimental conditions. The in vitro results indicated that BMP2 enhanced cell proliferation by 130%–157% and osteogenic differentiation by approximately two-fold in type 2 diabetic BMSCs. The expression levels of β-catenin, cyclin D1, Runx2 and c-myc related to the Wnt signaling pathway were also upregulated from 180% to 212% in BMP2-induced type 2 diabetic rat BMSCs, while the level of GSK3β decreased to 43%. In BMP2-induced type 2 diabetic BMSCs with calcium phosphate cement (CPC) scaffolds for osteoblast study in vivo, the appearance of newly formed bone dramatically increased to 175% compared with type 2 diabetic BMSCs. These data demonstrated that BMP2 enhanced bone regeneration in diabetic BMSCs by stimulating the Wnt signaling pathway with the accumulation of β-catenin and the depressed expression of GSK3β. Diabetic BMSCs associated with BMP2 might be a potential tissue-engineered construct for bone defects in type 2 diabetes mellitus.

Minipig-BMSCs Combined with a Self-Setting Calcium Phosphate Paste for Bone Tissue Engineering.

Calcium phosphate cements (CPCs) are a new generation of bone repair materials with good biocompatibility for various stem cells. The minipig is a recommended large animal model for bone engineering research. This study aimed to evaluate the feasibility of utilizing CPC scaffolds for the adhesion, proliferation, and osteogenic differentiation of minipig's bone marrow mesenchymal stem cells (pBMSCs). Passage 3 pBMSCs were seeded on the CPC scaffold and cultured with osteogenic culture medium (osteogenic group) or normal medium (control group). The density of viable cells increased in both groups, and pBMSCs firmly attached and spread well on the CPC scaffold. The alkaline phosphatase (ALP) activity in the osteogenic group had significantly increased on day 7 (D7) and peaked on D14. qRT-PCR revealed that mRNA levels of ALP and three osteogenic marker genes were significantly higher on D4, D7, and D14 in the osteogenic group. Alizarin Red S staining showed a significantly higher degree of bone mineralization from D7, D14 to D21 in the osteogenic group. These results indicated that pBMSCs can attach, proliferate well on CPC scaffold, and be successfully induced to differentiate into osteogenic cells. Our findings may be helpful for bone tissue engineering and the studies of bone regeneration.

PEGylated poly(glycerol sebacate)-modified calcium phosphate scaffolds with desirable mechanical behavior and enhanced osteogenic capacity.

Calcium phosphate (CaP) scaffolds have been widely used as bone graft substitutes, but undesirable mechanical robustness and bioactivity greatly hamper its availability in clinic application. To address these issues, PEGylated poly (glycerol sebacate) (PEGS), a hydrophilic elastomer, was used to modify a model calcium phosphate cement (CPC) scaffold for bone regeneration in this study. The PEGS pre-polymer with PEG content from 0% to 40% was synthesized and was subsequently coated onto the pre-fabricated CPC scaffolds by facile infiltration and thermal-crosslink process. Compression strength and toughness of the CPC/PEGS composite scaffold (defined as CPX/Y, X referred to the PEG content in PEGS and Y referred to PEGS amount in final scaffold) were effectively tailored with increasing coating amount and PEG content, and CPX/Y exhibited an optimal compressive strength of 3.82MPa and elongation at break of 13.20%, around 5-fold and 3-fold enhancement compared to the CPC. In vitro cell experiment with BMSCs as model indicated that coating and PEG-modified synchronously facilitated cell attachment and proliferation in a dose-dependent manner. Particularly, osteogenic differentiation of BMSCs on PEGS/CPC scaffold was strongly enhanced, especially for CP20/18. Further in vivo experiments confirmed that PEGS/CPC induced promoted osteogenesis in striking contrast to CPC and PGS/CPC. Collectively, hybrids scaffolds (around 18% coating amount and PEG content from 20% to 40%) with the combination of enhanced mechanical behavior and up-regulated cellular response were optimized and PEGS/CaP scaffolds can be deemed as a desirable option for bone tissue engineering. Insufficient mechanical robustness and bioactivity still limit the availability of calcium phosphate (CaP) scaffolds in clinic application. Herein, calcium phosphate cement (CPC) scaffold, as a model CaP-matrix material, was modified with PEGylated PGS (PEGS) polymers by facile infiltration and thermal-crosslink process. Such biomimetic combination of PEGS and CaP-matrix porous scaffold was first explored, without affecting its porous structure. In this study, CPC scaffold was endowed with robust mechanical behavior and promoted bioactivity by simultaneously optimizing the amount of polymer-coating and the PEG content in PGS. In rat critical-sized calvarial defects repairing, osteogenic efficacy of PEGS/CPC further demonstrated the potential for application in bone tissue regeneration. The design concept proposed in this study might provide new insights into the development of future tissue engineering materials.

Injectable calcium phosphate with hydrogel fibers encapsulating induced pluripotent, dental pulp and bone marrow stem cells for bone repair

Human induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs), dental pulp stem cells (hDPSCs) and bone marrow MSCs (hBMSCs) are exciting cell sources in regenerative medicine. However, there has been no report comparing hDPSCs, hBMSCs and hiPSC-MSCs for bone engineering in an injectable calcium phosphate cement (CPC) scaffold. The objectives of this study were to: (1) develop a novel injectable CPC containing hydrogel fibers encapsulating stem cells for bone engineering, and (2) compare cell viability, proliferation and osteogenic differentiation of hDPSCs, hiPSC-MSCs from bone marrow (BM-hiPSC-MSCs) and from foreskin (FS-hiPSC-MSCs), and hBMSCs in CPC for the first time. The results showed that the injection did not harm cell viability. The porosity of injectable CPC was 62%. All four types of cells proliferated and differentiated down the osteogenic lineage inside hydrogel fibers in CPC. hDPSCs, BM-hiPSC-MSCs, and hBMSCs exhibited high alkaline phosphatase, runt-related transcription factor, collagen I, and osteocalcin gene expressions. Cell-synthesized minerals increased with time (p<0.05), with no significant difference among hDPSCs, BM-hiPSC-MSCs and hBMSCs (p>0.1). Mineralization by hDPSCs, BM-hiPSC-MSCs, and hBMSCs inside CPC at 14d was 14-fold that at 1d. FS-hiPSC-MSCs were inferior in osteogenic differentiation compared to the other cells. In conclusion, hDPSCs, BM-hiPSC-MSCs and hBMSCs are similarly and highly promising for bone tissue engineering; however, FS-hiPSC-MSCs were relatively inferior in osteogenesis. The novel injectable CPC with cell-encapsulating hydrogel fibers may enhance bone regeneration in dental, craniofacial and orthopedic applications.

Design and Fabrication of Complex Scaffolds for Bone Defect Healing: Combined 3D Plotting of a Calcium Phosphate Cement and a Growth Factor-Loaded Hydrogel

Additive manufacturing enables the fabrication of scaffolds with defined architecture. Versatile printing technologies such as extrusion-based 3D plotting allow in addition the incorporation of biological components increasing the capability to restore functional tissues. We have recently described the fabrication of calcium phosphate cement (CPC) scaffolds by 3D plotting of an oil-based CPC paste under mild conditions. In the present study, we have developed a strategy for growth factor loading based on multichannel plotting: a biphasic scaffold design was realised combining CPC with VEGF-laden, highly concentrated hydrogel strands. As hydrogel component, alginate and an alginate-gellan gum blend were evaluated; the blend exhibited a more favourable VEGF release profile and was chosen for biphasic scaffold fabrication. After plotting, two-step post-processing was performed for both, hydrogel crosslinking and CPC setting, which was shown to be compatible with both materials. Finally, a scaffold was designed and fabricated which can be applied for testing in a rat critical size femur defect. Optimization of CPC plotting enabled the fabrication of highly resolved structures with strand diameters of only 200µm. Micro-computed tomography revealed a precise strand arrangement and an interconnected pore space within the biphasic scaffold even in swollen state of the hydrogel strands.

Fabrication of macroporous cement scaffolds using PEG particles: In vitro evaluation with induced pluripotent stem cell-derived mesenchymal progenitors

Calcium phosphate cements (CPCs) have been extensively used in reconstructive dentistry and orthopedics, but it is only recently that CPCs have been combined with stem cells to engineer biological substitutes with enhanced healing potential. In the present study, macroporous CPC scaffolds with defined composition were fabricated using an easily reproduced synthesis method, with minimal fabrication and processing steps. Scaffold pore size and porosity, essential for cell infiltration and tissue ingrowth, were tuned by varying the content and size of polyethylene glycol (PEG) particles, resulting in 9 groups with different architectural features. The scaffolds were characterized for chemical composition, porosity and mechanical properties, then tested in vitro with human mesenchymal progenitors derived from induced pluripotent stem cells (iPSC-MPs). Biomimetic decellularized bone scaffolds were used as reference material in this study. Our manufacturing process resulted in the formation of macroporous monetite scaffolds with no residual traces of PEG. The size and content of PEG particles was found to affect scaffold porosity, and thus mechanical properties. Irrespective of pore size and porosity, the CPC scaffolds fabricated in this study supported adhesion and viability of human iPSC-MPs similarly to decellularized bone scaffolds. However, the architectural features of the scaffolds were found to affect the expression of bone specific genes, suggesting that specific scaffold groups could be more suitable to direct human iPSC-MPs in vitro toward an osteoblastic phenotype. Our simplistic fabrication method allows rapid, inexpensive and reproducible construction of macroporous CPC scaffolds with tunable architecture for potential use in dental and orthopedic applications.

Graphene Oxide-Copper Nanocomposite-Coated Porous CaP Scaffold for Vascularized Bone Regeneration via Activation of Hif-1α.

Graphene has been studied for its in vitro osteoinductive capacity. However, the in vivo bone repair effects of graphene-based scaffolds remain unknown. The aqueous soluble graphene oxide-copper nanocomposites (GO-Cu) are fabricated, which are used to coat porous calcium phosphate (CaP) scaffolds for vascularized bone regeneration. The GO-Cu nanocomposites, containing crystallized CuO/Cu2 O nanoparticles of ≈30 nm diameters, distribute uniformly on the surfaces of the porous scaffolds and maintain a long-term release of Cu ions. In vitro, the GO-Cu coating enhances the adhesion and osteogenic differentiation of rat bone marrow stem cells (BMSCs). It is also found that by activating the Erk1/2 signaling pathway, the GO-Cu nanocomposites upregulate the expression of Hif-1α in BMSCs, resulting in the secretion of VEGF and BMP-2 proteins. When transplanted into rat with critical-sized calvarial defects, the GO-Cu-coated calcium phosphate cement (CPC) scaffolds (CPC/GO-Cu) significantly promote angiogenesis and osteogenesis. Moreover, it is observed via histological sections that the GO-Cu nanocomposites are phagocytosed by multinucleated giant cells. The results suggest that GO-Cu nanocomposite coatings can be utilized as an attractive strategy for vascularized bone regeneration.

Preparation and characterization of calcium phosphate/pectin scaffolds for bone tissue engineering

A calcium phosphate cement (CPC) scaffold has been used to repair bone defects, but its low compressive strength and poor osteogenesis greatly hinder its clinical application.

Fabrication of Poly-l-lactic Acid/Dicalcium Phosphate Dihydrate Composite Scaffolds with High Mechanical Strength-Implications for Bone Tissue Engineering.

Scaffolds were fabricated from poly-l-lactic acid (PLLA)/dicalcium phosphate dihydrate (DCPD) composite by indirect casting. Sodium citrate and PLLA were used to improve the mechanical properties of the DCPD scaffolds. The resulting PLLA/DCPD composite scaffold had increased diametral tensile strength and fracture energy when compared to DCPD only scaffolds (1.05 vs. 2.70 MPa and 2.53 vs. 12.67 N-mm, respectively). Sodium citrate alone accelerated the degradation rate by 1.5 times independent of PLLA. Cytocompatibility of all samples were evaluated using proliferation and differentiation parameters of dog-bone marrow stromal cells (dog-BMSCs). The results showed that viable dog-BMSCs attached well on both DCPD and PLLA/DCPD composite surfaces. In both DCPD and PLLA/DCPD conditioned medium, dog-BMSCs proliferated well and expressed alkaline phosphatase (ALP) activity indicating cell differentiation. These findings indicate that incorporating both sodium citrate and PLLA could effectively improve mechanical strength and biocompatibility without increasing the degradation time of calcium phosphate cement scaffolds for bone tissue engineering purposes.

The promotion of bone tissue regeneration by BMP2-derived peptide P24-loaded calcium phosphate cement microspheres

Calcium phosphate cement (CPC) scaffolds have been widely used as bone graft substitutes, but their clinical application is restricted by the undesirable osteoinductivity and slow degradability. We developed new CPC microspheres loaded with bone morphogenetic protein-2 (BMP-2)-derived peptide P24 (P24/CPC). The BMP2-derived peptide P24 was evenly pasted with CPC microspheres, which maintained the bioactivity of BMP-2. We evaluated the effect of the P24/CPC microspheres on osteogenesis and examined the underlying mechanism in vitro and in vivo. The in vitro studies showed that the bioactivity of P24 was preserved in the P24/CPC microspheres and the P24 release duration lasted up to 39 days. The P24/CPC microspheres with different proportions of P24 (2% and 4%) induced high proliferation of bone marrow stromal cells (BMSCs) and well supported cell adhesion on the sphere surfaces. Furthermore, the cells exhibited higher alkaline phosphatase activity and higher expression of osteogenic-specific genes (OCN and Runx2) in 4% P24/CPC than in the CPC and control groups (P<0.05). Therefore, these findings indicated the enhanced osteoblastic differentiation of BMSCs on P24/CPC microspheres. In vivo osteoinductive studies revealed that the degrees of ectopic osteogenesis in the dorsal muscle pocket of rat were significantly higher in the P24/CPC microspheres than in the CPC microspheres. Finally, the CPC or P24/CPC microspheres were implanted into rabbit femoral condyle defects and P24/CPC serial groups were observed for 4, 8, and 12 weeks. Computed tomography (CT) and histological and histomorphological studies proved that P24/CPC microspheres were biocompatible, bioabsorbable, and osteoinductive. In conclusion, this study demonstrated that P24/CPC microspheres can enhance osteogenesis in vitro and promote bone regeneration in vivo. Furthermore, our results confirmed that BMP2-derived peptide P24 can have a beneficial effect on the bone-generating properties of CPC microspheres, suggesting that P24/CPC microspheres are strong potential bone graft substitutes in bone tissue engineering.

3D plotting of growth factor loaded calcium phosphate cement scaffolds

Additive manufacturing allows to widely control the geometrical features of implants. Recently, we described the fabrication of calcium phosphate cement (CPC) scaffolds by 3D plotting of a storable CPC paste based on water-immiscible carrier liquid. Plotting and hardening is conducted under mild conditions allowing the (precise and local) integration of biological components. In this study, we have developed a procedure for efficient loading of growth factors in the CPC scaffolds during plotting and demonstrated the feasibility of this approach. Bovine serum albumin (BSA) or vascular endothelial growth factor (VEGF), used as model proteins, were encapsulated in chitosan/dextran sulphate microparticles which could be easily mixed into the CPC paste in freeze-dried state. In order to prevent leaching of the proteins during cement setting, usually carried out by immersion in aqueous solutions, the plotted scaffolds were aged in water-saturated atmosphere (humidity). Setting in humidity avoided early loss of loaded proteins but provided sufficient amount of water to allow cement setting, as indicated by XRD analysis and mechanical testing in comparison to scaffolds set in water. Moreover, humidity-set scaffolds were characterised by altered, even improved properties: no swelling or crack formation was observed and accordingly, surface topography, total porosity and compressive modulus of the humidity-set scaffolds differed from those of the water-set counterparts. Direct cultivation of mesenchymal stem cells on the humidity-set scaffolds over 21days revealed their cytocompatibility. Maintenance of the bioactivity of VEGF during the fabrication procedure was proven in indirect and direct culture experiments with endothelial cells. Statement of SignificanceAdditive manufacturing techniques allow the fabrication of implants with defined architecture (inner pore structure and outer shape). Especially printing technologies conducted under mild conditions allow additionally the (spatially controlled) integration of biological components such as drugs or growth factors. That enables the generation of individualized implants which can better meet the requirements of a patient and of tissue engineering constructs. To our knowledge, simultaneous printing of biological components was up to now only described for hydrogel/biopolymer-based materials which suffer from poor mechanical properties. In contrast, we have developed a procedure (based on 3D plotting of a calcium phosphate cement paste) for the fabrication of designed and growth factor loaded calcium-phosphate-based scaffolds applicable for bone regeneration.

High-Fat Diet/Low-Dose Streptozotocin-Induced Type 2 Diabetes in Rats Impacts Osteogenesis and Wnt Signaling in Bone Marrow Stromal Cells.

Bone regeneration disorders are a significant problem in patients with type 2 diabetes mellitus. Bone marrow stromal cells (BMSCs) are recognized as ideal seed cells for tissue engineering because they can stimulate osteogenesis during bone regeneration. Therefore, the aim of this study was to investigate the osteogenic potential of BMSCs derived from type 2 diabetic rats and the pathogenic characteristics of dysfunctional BMSCs that affect osteogenesis. BMSCs were isolated from normal and high-fat diet+streptozotocin-induced type 2 diabetic rats. Cell metabolic activity, alkaline phosphatase (ALP) activity, mineralization and osteogenic gene expression were reduced in the type 2 diabetic rat BMSCs. The expression levels of Wnt signaling genes, such as β-catenin, cyclin D1 and c-myc, were also significantly decreased in the type 2 diabetic rat BMSCs, but the expression of GSK3β remained unchanged. The derived BMSCs were cultured on calcium phosphate cement (CPC) scaffolds and placed subcutaneously into nude mice for eight weeks; they were detected at a low level in newly formed bone. The osteogenic potential of the type 2 diabetic rat BMSCs was not impaired by the culture environment, but it was impaired by inhibition of the Wnt signaling pathway, likely due to an insufficient accumulation of β-catenin rather than because of GSK3β stimulation. Using BMSCs derived from diabetic subjects could offer an alternative method of regenerating bone together with the use of supplementary growth factors to stimulate the Wnt signaling pathway.

Biphasic Calcium Phosphate Scaffold with Interconnected Pores Prepared by Sintering Calcium Phosphate Bone Cement

This proposal aims to develop a newly, stable, excellent and environmental process of manufacturing scaffolds with virtually identical biphasic calcium phosphate compositions. Calcium phosphate cements (CPCs), which combines calcium orthophosphate powders with a liquid leading to a paste that hardens spontaneously at low temperatures, have potential to be used as a porous template for dental bone grafting substitutes [1,2]. Such newly developed sintering processes having the bone grafts with properties of bioactivity or even bioresorbability would be applied in many clinical setting. Template materials combine calcium orthophosphate powders with a liquid leading to a paste that hardens spontaneously at low temperatures. Hence, CPCs could be applied as scaffolds to support cell/tissue growth [3, 4]. This paper studies CPC scaffolds processing by foaming cement's paste state in which was added phasic stabilizer of magnesia and foaming agent of sucrose. The X-ray diffraction was performed to identify the phases of bone grafting substitutes, and we also used scanning electron microscope to observe the structure and pores of bone grafting substitutes. The cell viability about biocompatibility of developed bone grafting substitutes was examined. The results showed that our bone grafting substitutes produced steady final biphasic products consisting of hydroxyapatite (HA) and beta-tricalcium phosphates (β-TCP). We observed interconnected pores and highly porosity in microstructure of the bone grafting substitutes. The cell viability was over 70 % to make sure that the bone grafting substitutes has excellent biocompatibility. In conclusion, using the slurry of calcium phosphate cements (CPCs) and pores forming agent set into a porous template would be a useful process for manufacturing bone graft substitutes.

Bone tissue engineering via human induced pluripotent, umbilical cord and bone marrow mesenchymal stem cells in rat cranium

Human induced pluripotent stem cells (hiPSCs) are an exciting cell source with great potential for tissue engineering. Human bone marrow mesenchymal stem cells (hBMSCs) have been used in clinics but are limited by several disadvantages, hence alternative sources of MSCs such as umbilical cord MSCs (hUCMSCs) are being investigated. However, there has been no report comparing hiPSCs, hUCMSCs and hBMSCs for bone regeneration. The objectives of this pilot study were to investigate hiPSCs, hUCMSCs and hBMSCs for bone tissue engineering, and compare their bone regeneration via seeding on biofunctionalized macroporous calcium phosphate cement (CPC) in rat cranial defects. For all three types of cells, approximately 90% of the cells remained alive on CPC scaffolds. Osteogenic genes were up-regulated, and mineral synthesis by cells increased with time in vitro for all three types of cells. The new bone area fractions at 12weeks (mean±sd; n=6) were (30.4±5.8)%, (27.4±9.7)% and (22.6±4.7)% in hiPSC–MSC–CPC, hUCMSC–CPC and hBMSC–CPC respectively, compared to (11.0±6.3)% for control (p<0.05). No significant differences were detected among the three types of stem cells (p>0.1). New blood vessel density was higher in cell-seeded groups than control (p<0.05). De novo bone formation and participation by implanted cells was confirmed via immunohistochemical staining. In conclusion, (1) hiPSCs, hUCMSCs and hBMSCs greatly enhanced bone regeneration, more than doubling the new bone amount of cell-free CPC control; (2) hiPSC–MSCs and hUCMSCs represented viable alternatives to hBMSCs; (3) biofunctionalized macroporous CPC-stem cell constructs had a robust capacity for bone regeneration.

Mechanical Activation Reinforced Porous Calcium Phosphate Cement

Calcium phosphate cement (CPC) powder was activated by ball milling to improve the mechanical properties of porous CPC scaffolds. The mechanical activation mechanism was investigated by specific surface analyses, X-ray diffraction (XRD) and scanning electron microscopy (SEM). After ball milling, the average particle sizes of BCPC powder decreased while the specific surface area, apparent density, bulk density, and compact density increased when compared with non-activated CPC powder. The porosity and compressive strength of porous CPC scaffolds prepared from ball-milled powders (BCPC-S) were (77.98 ± 0.58)% and (4.11 ± 0.46) MPa, both significantly higher than those non-activated CPC powders (CPC-S), whose porosity and compressive strength were (64.23  2.32)% and (1.99 ± 0.43) MPa, respectively. SEM revealed that there were two types of pores in the BCPC-S: one ranged a few microns in size and the other ranged several hundred microns. XRD indicated that grain sizes and crystallinities of dicalcium phosphate dehydrate (DCPD), α-tricalcium phosphate (α-TCP), calcium carbonate (CaCO3) and hydroxyapatite (HA) in BCPC powder decreased, due to the mechanical activation compared to 第 4期 黄 萍, 等: 机械活化增强多孔磷酸钙骨水泥支架的研究 433 those of the non-activated CPC powders. In addition, the mechanical activation resulted in the conversion of DCPD to dicalcium phosphate anhydrous (DCPA), which promoted the hydration of CPC and the precipitation of HA, and improved the compressive strength of BCPC-S finally. This study provided a potential approach to improve the mechanical properties of porous CaP based scaffold to meet the clinic requirement.

Effect of NELL1 gene overexpression in iPSC-MSCs seeded on calcium phosphate cement

Human induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) are a promising source of patient-specific stem cells with great regenerative potential. There has been no report on NEL-like protein 1 (NELL1) gene modification of iPSC-MSCs. The objectives of this study were to genetically modify iPSC-MSCs with NELL1 overexpression for bone tissue engineering, and investigate the osteogenic differentiation of NELL1 gene-modified iPSC-MSCs seeded on Arg-Gly-Asp (RGD)-grafted calcium phosphate cement (CPC) scaffold. Cells were transduced with red fluorescence protein (RFP-iPSC-MSCs) or NELL1 (NELL1-iPSC-MSCs) by a lentiviral vector. Cell proliferation on RGD-grafted CPC scaffold, osteogenic differentiation and bone mineral synthesis were evaluated. RFP-iPSC-MSCs stably expressed high levels of RFP. Both the NELL1 gene and NELL1 protein levels were confirmed higher in NELL1-iPSC-MSCs than in RFP-iPSC-MSCs using RT-PCR and Western blot (P<0.05). Alkaline phosphatase activity was increased by 130% by NELL1 overexpression at 14days (P<0.05), indicating that NELL1 promoted iPSC-MSC osteogenic differentiation. When seeded on RGD-grafted CPC, NELL1-iPSC-MSCs attached and expanded similarly well to RFP-iPSC-MSCs. At 14days, the runt-related transcription factor 2 (RUNX2) gene level of NELL1-iPSC-MSCs was 2.0-fold that of RFP-iPSC-MSCs. The osteocalcin (OC) level of NELL1-iPSC-MSCs was 3.1-fold that of RFP-iPSC-MSCs (P<0.05). The collagen type I alpha 1 (COL1A1) gene level of NELL1-iPSC-MSCs was 1.7-fold that of RFP-iPSC-MSCs at 7days (P<0.05). Mineral synthesis was increased by 81% in NELL1-iPSC-MSCs at 21days. In conclusion, NELL1 overexpression greatly enhanced the osteogenic differentiation and mineral synthesis of iPSC-MSCs on RGD-grafted CPC scaffold for the first time. The novel NELL1-iPSC-MSC seeded RGD-CPC construct is promising for enhancing bone engineering.

Enhanced healing of rabbit segmental radius defects with surface-coated calcium phosphate cement/bone morphogenetic protein-2 scaffolds

Large osseous defects remain a difficult clinical problem in orthopedic surgery owing to the limited effective therapeutic options, and bone morphogenetic protein-2 (BMP-2) is useful for its potent osteoinductive properties in bone regeneration. Here we build a strategy to achieve prolonged duration time and help inducting new bone formation by using water-soluble polymers as a protective film. In this study, calcium phosphate cement (CPC) scaffolds were prepared as the matrix and combined with sodium carboxymethyl cellulose (CMC-Na), hydroxypropylmethyl cellulose (HPMC), and polyvinyl alcohol (PVA) respectively to protect from the digestion of rhBMP-2. After being implanted in the mouse thigh muscles, the surface-modified composite scaffolds evidently induced ectopic bone formation. In addition, we further evaluated the in vivo effects of surface-modified scaffolds in a rabbit radius critical defect by radiography, three dimensional micro-computed tomographic (μCT) imaging, synchrotron radiation-based micro-computed tomographic (SRμCT) imaging, histological analysis, and biomechanical measurement. The HPMC-modified CPC scaffold was regarded as the best combination for segmental bone regeneration in rabbit radius.

Human embryonic stem cells and macroporous calcium phosphate construct for bone regeneration in cranial defects in rats

Human embryonic stem cells (hESCs) are an exciting cell source as they offer an unlimited supply of cells that can differentiate into all cell types for regenerative medicine applications. To date, there has been no report on hESCs with calcium phosphate cement (CPC) scaffolds for bone regeneration in vivo. The objectives of this study were to: (i) investigate hESCs for bone regeneration in vivo in critical-sized cranial defects in rats; and (ii) determine the effects of cell seeding and platelets in macroporous CPC on new bone and blood vessel formation. hESCs were cultured to yield mesenchymal stem cells (MSCs), which underwent osteogenic differentiation. Four groups were tested in rats: (i) CPC control without cells; (ii) CPC with hESC-derived MSCs (CPC+hESC-MSC); (iii) CPC with hESC-MSCs and 30% human platelet concentrate (hPC) (CPC+hESC-MSC+30% hPC); and (iv) CPC+hESC-MSC+50% hPC. In vitro, MSCs were derived from embryoid bodies of hESCs. Cells on CPC were differentiated into the osteogenic lineage, with highly elevated alkaline phosphatase and osteocalcin expressions, as well as mineralization. At 12weeks in vivo, the groups with hESC-MSCs and hPC had three times as much new bone as, and twice the blood vessel density of, the CPC control. The new bone in the defects contained osteocytes and blood vessels, and the new bone front was lined with osteoblasts. The group with 30% hPC and hESC-MSCs had a blood vessel density that was 49% greater than the hESC-MSC group without hPC, likely due to the various growth factors in the platelets enhancing both new bone and blood vessel formation. In conclusion, hESCs are promising for bone tissue engineering, and hPC can enhance new bone and blood vessel formation. Macroporous CPC with hESC-MSCs and hPC may be useful for bone regeneration in craniofacial and orthopedic applications.

Stem Cells and Calcium Phosphate Cement Scaffolds for Bone Regeneration

Calcium phosphate cements (CPCs) have excellent biocompatibility and osteoconductivity for dental, craniofacial, and orthopedic applications. This article reviews recent developments in stem cell delivery via CPC for bone regeneration. This includes: (1) biofunctionalization of the CPC scaffold, (2) co-culturing of osteoblasts/endothelial cells and prevascularization of CPC, (3) seeding of CPC with different stem cell species, (4) human umbilical cord mesenchymal stem cell (hUCMSC) and bone marrow MSC (hBMSC) seeding on CPC for bone regeneration, and (5) human embryonic stem cell (hESC) and induced pluripotent stem cell (hiPSC) seeding with CPC for bone regeneration. Cells exhibited good attachment/proliferation in CPC scaffolds. Stem-cell-CPC constructs generated more new bone and blood vessels in vivo than did the CPC control without cells. hUCMSCs, hESC-MSCs, and hiPSC-MSCs in CPC generated new bone and blood vessels similar to those of hBMSCs; hence, they were viable cell sources for bone engineering. CPC with hESC-MSCs and hiPSC-MSCs generated new bone two- to three-fold that of the CPC control. Therefore, this article demonstrates that: (1) CPC scaffolds are suitable for delivering cells; (2) hUCMSCs, hESCs, and hiPSCs are promising alternatives to hBMSCs, which require invasive procedures to harvest with limited cell quantity; and (3) stem-cell-CPC constructs are highly promising for bone regeneration in dental, craniofacial, and orthopedic applications.