Macroporous Calcium Phosphate Research Articles

Mechanical Properties of Graded Macroporous Calcium Phosphate Ceramics of Tailored Architecture

Mechanical Properties of Graded Macroporous Calcium Phosphate Ceramics of Tailored Architecture

Fabrication of Radio-Opaque and Macroporous Injectable Calcium Phosphate Cement.

The aim of this work was the development of injectable radio-opaque and macroporous calcium phosphate cement (CPC) to be used as a bone substitute for the treatment of pathologic vertebral fractures. A CPC was first rendered radio-opaque by the incorporation of zirconium dioxide (ZrO2). In order to create macroporosity, poly lactic-co-glycolic acid (PLGA) microspheres around 100 μm were homogeneously incorporated into the CPC as observed by scanning electron microscopy. Physicochemical analyses by X-ray diffraction and Fourier transform infrared spectroscopy confirmed the brushite phase of the cement. The mechanical properties of the CPC/PLGA cement containing 30% PLGA (wt/wt) were characterized by a compressive strength of 2 MPa and a Young's modulus of 1 GPa. The CPC/PLGA exhibited initial and final setting times of 7 and 12 min, respectively. Although the incorporation of PLGA microspheres increased the force necessary to inject the cement and decreased the percentage of injected mass as a function of time, the CPC/PLGA appeared fully injectable at 4 min. Moreover, in comparison with CPC, CPC/PLGA showed a full degradation in 6 weeks (with 100% mass loss), and this was associated with an acidification of the medium containing the CPC/PLGA sample (pH of 3.5 after 6 weeks). A cell viability test validated CPC/PLGA biocompatibility, and in vivo analyses using a bone defect assay in the caudal vertebrae of Wistar rats showed the good opacity of the CPC through the tail and a significant increased degradation of the CPC/PLGA cement a month after implantation. In conclusion, this injectable CPC scaffold appears to be an interesting material for bone substitution.

Treatment of Critical Size Femoral Bone Defects with Biomimetic Hybrid Scaffolds of 3D Plotted Calcium Phosphate Cement and Mineralized Collagen Matrix.

To treat critical-size bone defects, composite materials and tissue-engineered bone grafts play important roles in bone repair materials. The purpose of this study was to investigate the bone regenerative potential of hybrid scaffolds consisting of macroporous calcium phosphate cement (CPC) and microporous mineralized collagen matrix (MCM). Hybrid scaffolds were synthetized by 3D plotting CPC and then filling with MCM (MCM-CPC group) and implanted into a 5 mm critical size femoral defect in rats. Defects left empty (control group) as well as defects treated with scaffolds made of CPC only (CPC group) and MCM only (MCM group) served as controls. Eight weeks after surgery, micro-computed tomography scans and histological analysis were performed to analyze the newly formed bone, the degree of defect healing and the activity of osteoclasts. Mechanical stability was tested by 3-point-bending of the explanted femora. Compared with the other groups, more newly formed bone was found within MCM-CPC scaffolds. The new bone tissue had a clamp-like structure which was fully connected to the hybrid scaffolds and thereby enhanced the biomechanical strength. Together, the biomimetic hybrid MCM-CPC scaffolds enhanced bone defect healing by improved osseointegration and their differentiated degradation provides spatial effects in the process of critical-bone defect healing.

Biomimetic versus sintered macroporous calcium phosphate scaffolds enhanced bone regeneration and human mesenchymal stromal cell engraftment in calvarial defects

In contrast to sintered calcium phosphates (CaPs) commonly employed as scaffolds to deliver mesenchymal stromal cells (MSCs) targeting bone repair, low temperature setting conditions of calcium deficient hydroxyapatite (CDHA) yield biomimetic topology with high specific surface area. In this study, the healing capacity of CDHA administering MSCs to bone defects is evaluated for the first time and compared with sintered beta-tricalcium phosphate (β-TCP) constructs sharing the same interconnected macroporosity. Xeno-free expanded human bone marrow MSCs attached to the surface of the hydrophobic β-TCP constructs, while infiltrating the pores of the hydrophilic CDHA. Implantation of MSCs on CaPs for 8 weeks in calvaria defects of nude mice exhibited complete healing, with bone formation aligned along the periphery of β-TCP, and conversely distributed within the pores of CDHA. Human monocyte-osteoclast differentiation was inhibited in vitro by direct culture on CDHA compared to β-TCP biomaterials and indirectly by administration of MSC-conditioned media generated on CDHA, while MSCs increased osteoclastogenesis in both CaPs in vivo. MSC engraftment was significantly higher in CDHA constructs, and also correlated positively with bone in-growth in scaffolds. These findings demonstrate that biomimetic CDHA are favorable carriers for MSC therapies and should be explored further towards clinical bone regeneration strategies. Statement of significanceDelivery of mesenchymal stromal cells (MSCs) on calcium phosphate (CaP) biomaterials enhances reconstruction of bone defects. Traditional CaPs are produced at high temperature, but calcium deficient hydroxyapatite (CDHA) prepared at room temperature yields a surface structure more similar to native bone mineral. The objective of this study was to compare the capacity of biomimetic CDHA scaffolds with sintered β-TCP scaffolds for bone repair mediated by MSCs for the first time. In vitro, greater cell infiltration occurred in CDHA scaffolds and following 8 weeks in vivo, MSC engraftment was higher in CDHA compared to β-TCP, as was bone in-growth. These findings demonstrate the impact of material features such as surface structure, and highlight that CDHA should be explored towards clinical bone regeneration strategies.

Histological Assessment of Injectable Macro-porous Calcium Phosphate Cement (CPC) Versus Autogenous Bone Graft on Healing of Osseous Defect (Experimental Animal Study)

Objective: The aim of this study is to evaluate histologically the healing potential of injectable macro-porous Calcium Phosphate Cement and autogenous bone. (Normal bone healing) on surgically induced bone defect on femur bone of rabbit. Materials and Methods: Twelve adult white male New Zealand rabbits were selected for this study. Rabbits were divided into two groups and the two groups according to a two evaluation periods divided into 2 subgroups. Rabbits were anesthetized with intramuscular anethesia, and two osseous defects had been created in the distal aspect of the femur bone.one of this defects injected by a grafting material (SI-HPMC CPC) and the other left empty as control (normal healing).Animal were sacrificed after two sacrifice dates and the femur bone excised for histological and statistical evaluation.esults: Rabbits from each group were sacrificed after 2 and 4 weeks. The femur bone containing the induced defect was dissected. Each femur bone was excised using hard tissue microtome. Each slice was then fixed in 10% neutral buffered formalin. The formalin-fixed bone samples were decalcified in 15% buffered formic acid solution and processed for routine histological examination using hematoxylin and eosin stain under light microscope.. Conclusion: According to our results in the present study, we can consider Si-HPMC CPC a viable alternative to the autogenous bone in the healing of osseous bone defects . In the present study, the new and simple method to prepare macroporous CPCs using Si-HPMC a foaming agent in connected syringe , result

Macroporous Calcium Phosphate Matrices Prepared Using the Technology of Self-Setting Cements

Macroporous Calcium Phosphate Matrices Prepared Using the Technology of Self-Setting Cements

Relationship between the Porous Structure Hierarchy and the Physical and Mechanical Properties of Calcium Phosphate Drug Carriers

A relationship between the structural-morphological and physical-mechanical properties of macro-porous calcium phosphate (CaP) coatings on the surface of titanium implants is studied. The produced CaP coatings can be used as carriers for target drug delivery in biomedical applications. The regularities of the formation of bulk (thickness of 15–130 μm) and rough (Ra = 1.7–8.5 μm) CaP coatings deposited by the micro-arc oxidation (MAO) method are studied. The coatings have a hierarchical porous structure and a uniform distribution of the structural elements (spheres and pores) over the surface. Correlations between the value of the applied voltage and structural-morphological and physical-mechanical characteristics of the CaP coatings have been established. The value of the coating adhesion strength has been determined with tensile tests. Both cohesive and mixed adhesive-cohesive pull-off of the MAO coating from the substrate have been identified.

Digital Light Processing of Freeze-cast Ceramic Layers for Macroporous Calcium Phosphate Scaffolds with Tailored Microporous Frameworks.

The objective of the present study is to demonstrate the versatility of the digital light processing (DLP) technique particularly when using a freeze-cast ceramic layer as the feedstock, which can manufacture porous calcium phosphate (CaP) scaffolds with arbitrarily designed macroporous structures with tailored microporous frameworks specially designed for bone scaffold applications. For this goal, we employed camphene-camphor as the freezing vehicle and porogen for the preparation of photocurable CaP suspensions containing diurethane dimethacrylate (UDMA) monomers. After freeze-casting, the CaP suspensions could be solidified at controlled temperatures (~33–38 °C) and then be photopolymerized by DLP. All produced CaP scaffolds fairly resembled the designed macroporous structures (the gyroid structure with two interpenetrating macropore networks). In addition, numerous micropores were created in the CaP filaments, while the microporosity increased with increasing the camphene-camphor amount from 40 vol % to 60 vol %. As a consequence, compressive strength and modulus of hierarchically porous CaP scaffolds decreased due to an increase in overall porosity. However, reasonable mechanical properties could be obtained at high porosities owing to the CaP frameworks constructed in a periodic manner. In addition, excellent water penetration capability, biocompatibility, and apatite-forming ability were obtained, which were attributed to the microporous CaP frameworks with good pore interconnectivity and large surface area.

Comparative evaluation of the osteogenic capacity of human mesenchymal stem cells from bone marrow and umbilical cord tissue.

Mesenchymal stem cells (MSCs) have been extensively investigated in the field of regenerative medicine. Human bone MSCs (BMSCs) have become a common type of seed cell for bone tissue engineering. However, the viability and cell number of BMSCs are negatively correlated with donor age, and as the extraction process is painful, this method has not been widely used. As human umbilical cord MSCs (UCMSCs) may be harvested inexpensively and inexhaustibly, the present study evaluated and compared the regenerative potential of UCMSCs and BMSCs to determine whether UCMSCs may be used as a novel cell type for bone regeneration. In the present study, the proliferation and osteogenic capacity of BMSCs and UCMSCs was compared in vitro. BMSCs and UCMSCs were respectively combined with biofunctionalized macroporous calcium phosphate cement, and their bone regenerative potentials were determined by investigating their capacity for ectopic bone formation in a nude mouse model as well as their efficacy in a rat model of tibia bone defect. The extent of bone regeneration was examined by X-ray, histological and immunohistochemical analyses. The results revealed that UCMSCs exhibited a good osteogenic differentiation potential, similarly to that of BMSCs, and that UCMSCs were able to contribute to the regeneration of bone and blood vessels. Furthermore, no significant differences were identified between BMSCs and UCMSCs in terms of their bone regenerative effect.

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.

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.

Blooming gelatin: an individual additive for enhancing nanoapatite precipitation, physical properties, and osteoblastic responses of nanostructured macroporous calcium phosphate bone cements.

In recent years, there has been a great interest in using natural polymers in the composition of calcium phosphate bone cements to enhance their physical, mechanical, and biological performance. Gelatin is a partially hydrolyzed form of collagen, a natural component of bone matrix. In this study, the effect of blooming gelatin on the nanohydroxyapatite precipitation, physical and mechanical properties, and cellular responses of a calcium phosphate bone cement (CPC) was investigated. Various concentrations of blooming gelatin (2, 5, and 8 wt.%) were used as the cement liquid and an equimolar mixture of tetracalcium phosphate and dicalcium phosphate was used as solid phase. The CPC without any gelatin additive was also evaluated as a control group. The results showed that gelatin accelerated hydraulic reactions of the cement paste, in which the reactants were immediately converted into nanostructured apatite precipitates after hardening. Gelatin molecules induced 4%–10% macropores (10–300 μm) into the cement structure, decreased initial setting time by ~190%, and improved mechanical strength of the as-set cement. Variation in the above-mentioned properties was influenced by the gelatin concentration and progressed with increasing the gelatin content. The numbers of the G-292 osteoblastic cells on gelatin-containing CPCs were higher than the control group at entire culture times (1–14 days), meanwhile better alkaline phosphatase (ALP) activity was determined using blooming gelatin additive. The observation of cell morphologies on the cement surfaces revealed an appropriate cell attachment with extended cell membranes on the cements. Overall, adding gelatin to the composition of CPC improved the handling characteristics such as setting time and mechanical properties, enhanced nanoapatite precipitation, and augmented the early cell proliferation rate and ALP activity.

Investigation of Macroporous Calcium Phosphate Cement Obtained by Foamed Gelatin Polymer

This study deals with the effect of gelatin on physical and mechanical properties of calcium phosphate bone cements. The mixture of tetracalcium phosphate (TTCP) and dicalcium phosphate (DCPA) as the cement powder was mixed with 6 wt% Na2HPO4 solution containing different amount (0, 2, 5 and 8% in w/w) of foamed gelatin as liquid phase. The physical properties were determined in the terms of setting time and macroporosity. The compressive strength was also checked before and after soaking the cements in simulated body fluid (SBF). The phase composition, microstructure and chemical groups were respectively determined using X-ray diffraction (XRD), Scanning electron microscopy (SEM) and Fourier transformation Infrared Spectroscopy (FTIR). The results showed that gelatin accelerated hydroxyapatite (HA) precipitation during setting and reduced the initial setting time from 35 min for cement without gelatin to 23 min for cement with the most amount of gelatin. Moreover, 17% (in v/v) macroporosity was induced in the cement structure using 8% solution of gelatin as the cement liquid. Gelatin addition promoted compressive strength of the set cement from 1.13 MPa (for gelatin-free cement) to 5.8 MPa.

&lt;i&gt;In Vitro&lt;/i&gt; Bioactivity of Macroporous Calcium Phosphate Scaffold for Biomedical Application

In this study, the effects of macroporous calcium phosphate (MCP) scaffold on bioactivity as an in-vitro model has been investigated. MCP scaffold was prepared using foam replication technique by combination of ceramic and polyurethane (PU). MCP was examined by scanning electron microscope (SEM) and X-ray diffractometer (XRD) to confirm its microstructure and phase composition respectively. Bioactivity in simulated body fluid (SBF) was characterized by apatite mineralisation on MCP scaffold surface, pH, calcium (Ca2+) and phosphate (PO43-) ion dissolution and compressive strength using universal testing machine (UTM). Experimental results showed the formation of apatite around the MCP scaffold after 30 days in SBF. pH value was gradually decreased up to 7 days and constant until 30 days. Dissolution of Ca2+ and PO43- ion in SBF due to the occurrence of MCP degradation, hence the compressive strength are gradually decreased gradually in line with immersion period. This material may be promising for biomedical application.

Influence of micro- and nanostructure on the osteogenic potency of macroporous calcium phosphate scaffolds

Influence of micro- and nanostructure on the osteogenic potency of macroporous calcium phosphate scaffolds

A simple and effective approach to prepare injectable macroporous calcium phosphate cement for bone repair: Syringe-foaming using a viscous hydrophilic polymeric solution

In this study, we propose a simple and effective strategy to prepare injectable macroporous calcium phosphate cements (CPCs) by syringe-foaming via hydrophilic viscous polymeric solution, such as using silanized-hydroxypropyl methylcellulose (Si-HPMC) as a foaming agent. The Si-HPMC foamed CPCs demonstrate excellent handling properties such as injectability and cohesion. After hardening the foamed CPCs possess hierarchical macropores and their mechanical properties (Young’s modulus and compressive strength) are comparable to those of cancellous bone. Moreover, a preliminary in vivo study in the distal femoral sites of rabbits was conducted to evaluate the biofunctionality of this injectable macroporous CPC. The evidence of newly formed bone in the central zone of implantation site indicates the feasibility and effectiveness of this foaming strategy that will have to be optimized by further extensive animal experiments. Statement of significanceA major challenge in the design of biomaterial-based injectable bone substitutes is the development of cohesive, macroporous and self-setting calcium phosphate cement (CPC) that enables rapid cell invasion with adequate initial mechanical properties without the use of complex processing and additives. Thus, we propose a simple and effective strategy to prepare injectable macroporous CPCs through syringe-foaming using a hydrophilic viscous polymeric solution (silanized-hydroxypropyl methylcellulose, Si-HPMC) as a foaming agent, that simultaneously meets all the aforementioned aims. Evidence from our in vivo studies shows the existence of newly formed bone within the implantation site, indicating the feasibility and effectiveness of this foaming strategy, which could be used in various CPC systems using other hydrophilic viscous polymeric solutions.

Setting reaction of α-TCP spheres and an acidic calcium phosphate solution for the fabrication of fully interconnected macroporous calcium phosphate

Interconnected macroporous calcium phosphate has attracted attention as an artificial bone substitute as well as a scaffold for tissue engineering. In this investigation, a setting reaction between α-tricalcium phosphate (α-TCP) spheres and an acidic calcium phosphate solution (0.2mol/L monocalcium phosphate monohydrate – 0.1mol/L phosphoric acid) was studied to assess the feasibility of fabricating fully interconnected macroporous calcium phosphate. When 1.3mm-diameter α-TCP spheres were exposed to an acidic calcium phosphate solution, brushite (dicalcium phosphate dihydrate) was formed on the surface of the α-TCP spheres. The precipitated brushite crystals interlocked with one another and bridged the α-TCP spheres, resulting in a 10min setting reaction at 37°C that produced fully interconnected macroporous calcium phosphate. The resultant calcium phosphate macroporous structure had a porosity of 49.7±2.5% and an average pore size of 312±160μm.

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.

Macro-porous calcium phosphate scaffold with collagen and growth factors for periodontal bone regeneration in dogs

The periodontal bone regeneration has always been considered as a major challenge in maxillofacial clinic. Unfortunately, current treatments cannot achieve optimal therapeutic effect. Self-setting calcium phosphate bone cement (CPC) is regarded as promising material to solve periodontal problem due to its good biocompatibility, mineral similarity to the natural bone’s composition. Moreover, it can be arbitrarily shaped, form bio-resorbable hydroxyapatite (HA) in situ to fill complex periodontal bone cavities perfectly. So far, there are lots of related in-vitro investigations; however, in-vivo experiments which could precisely show the real effects of CPC’s internal performances are rarely reported. Therefore, in this study, CPC were implanted into bilateral mandible defect of 8 beagles to evaluate its degradation ability and osteogenesis through Micro-CT scan and histological morphology analysis at determined time. Because of the intrinsic drawbacks of raw CPC, reinforced ingredients including collagen and growth factors were selected to synthesis different modified-CPC systems. The results illustrated that the samples’ degradation ability could be largely increased by nearly 60% with the help of macro-porous structure and collagen. In addition, by using the same ingredients, the osteogenisis of those samples could be promoted by 12%. What’s more, growth factors were proved to be the most important factor to increase the CPC’s new bone formation ability (22% more new bone could be obtained after using growth factors after 6 months implantation). The data obtained from histological analysis has presented the similar changing trend. Overall, these results illustrated macro-porous structure; collagen and growth factors could both effectively promote the CPC’s degradation ability and osteogenesis in vivo, which suggested the macro-porous CPC with collagen and growth factors would be promising as bioactive materials for periodontal bone tissue regeneration.

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.