Macroporous Calcium Phosphate Cement Research Articles

Incorporation of synthetic water-soluble curcumin polymeric drug within calcium phosphate cements for bone defect repairing.

Modified macroporous structures and active osteogenic substances are necessary to overcome the limited bone regeneration capacity and low degradability of self-curing calcium phosphate cement (CPC). Curcumin (CUR), which possesses strong osteogenic activity and poor aqueous solubility/bioavailability, esterifies the side chains in hyaluronic acid (HA) to form a water-soluble CUR-HA macromolecule. In this study, we incorporated the CUR-HA and glucose microparticles (GMPs) into the CPC powder to fabricate the CUR-HA/GMP/CPC composite, which not only retained the good injectability and mechanical strength of bone cements, but also significantly increased the cement porosity and sustained release property of CUR-HA in vitro. CUR-HA incorporation greatly improved the differentiation ability of bone marrow mesenchymal stem cells (BMSCs) to osteoblasts by activating the RUNX family transcription factor 2/fibroblast growth factor 18 (RUNX2/FGF18) signaling pathway, increasing the expression of osteocalcin and enhancing the alkaline phosphatase activity. In addition, in vivo implantation of CUR-HA/GMP/CPC into femoral condyle defects dramatically accelerated the degradation rate of cement and boosted local vascularization and osteopontin protein expression, and consequently promoted rapid bone regeneration. Therefore, macroporous CPC based composite cement with CUR-HA shows a remarkable ability to repair bone defects and is a promising translational application of modified CPC in clinical practice.

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.

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

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.

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.

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.

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.

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.

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.

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.

Umbilical cord and bone marrow mesenchymal stem cell seeding on macroporous calcium phosphate for bone regeneration in rat cranial defects

Human umbilical cord mesenchymal stem cells (hUCMSCs) are inexhaustible and can be harvested at a low cost without an invasive procedure. However, there has been no report on comparing hUCMSCs with human bone marrow MSCs (hBMSCs) for bone regeneration in vivo. The aim of this study was to investigate hUCMSC and hBMSC seeding on macroporous calcium phosphate cement (CPC), and to compare their bone regeneration in critical-sized cranial defects in rats. Cell attachment, osteogenic differentiation and mineral synthesis on RGD-modified macroporous CPC were investigated in vitro. Scaffolds with cells were implanted in 8-mm defects of athymic rats. Bone regeneration was investigated via micro-CT and histological analysis at 4, 12, and 24 weeks. Three groups were tested: CPC with hUCMSCs, CPC with hBMSCs, and CPC control without cells. Percentage of live cells and cell density on CPC in vitro were similarly good for hUCMSCs and hBMSCs. Both cells had high osteogenic expressions of alkaline phosphatase, osteocalcin, collagen I, and Runx2. Bone mineral density and trabecular thickness in hUCMSC and hBMSC groups in vivo were greater than those of CPC control group. New bone amount for hUCMSC-CPC and hBMSC-CPC constructs was increased by 57% and 88%, respectively, while blood vessel density was increased by 15% and 20%, than CPC control group at 24 weeks. hUCMSC-CPC and hBMSC-CPC groups generally had statistically similar bone mineral density, new bone amount and vessel density. In conclusion, hUCMSCs seeded on CPC were shown to match the bone regeneration efficacy of hBMSCs in vivo for the first time. Both hUCMSC-CPC and hBMSC-CPC constructs generated much more new bone and blood vessels than CPC without cells. Macroporous RGD-grafted CPC with stem cell seeding is promising for craniofacial and orthopedic repairs.

Preparation of Hierarchically Porous MBG/CPC Scaffold and Its Performance on Drug Loading and Release

Hierarchically composite scaffold(MBG/CPC) was successfully prepared by loading of mesoporous bioactive glasses(MBG) into macroporous calcium phosphate cement(CPC) with simply centrifuging method.It is revealed that MBG loading capacity on CPC and the BET surface area of MBG/CPC can reach 47.2% and 100.1 m2/g,respectively.In contrast,almost no MBG loading is detected by using traditional impregnation route.Drug loading and releasing tests show that DOX loading capacity on hierarchically composite scaffold MBG/CPC is 46 mg/g,which is 11.5 times of that on pure CPC.Importantly,a sustained drug release in vitro performance is presented on MBG/CPC.

Prevascularization of a Gas-Foaming Macroporous Calcium Phosphate Cement Scaffold Via Coculture of Human Umbilical Vein Endothelial Cells and Osteoblasts

The lack of a vasculature in tissue-engineered constructs is currently a major challenge in tissue regeneration. There has been no report of prevascularization of macroporous calcium phosphate cement (CPC) via coculture of endothelial cells and osteoblasts. The objectives of this study were to (1) investigate coculture of human umbilical vein endothelial cells (HUVEC) and human osteoblasts (HOB) on macroporous CPC for the first time; and (2) develop a new microvasculature-CPC construct with angiogenic and osteogenic potential. A gas-foaming method was used to create macropores in CPC. HUVEC and HOB were seeded with a ratio of HUVEC:HOB=4:1, at 1.5×10(5) cells/scaffold. The constructs were cultured for up to 42 days. CPC with a porosity of 83% had a flexural strength (mean±SD; n=6) of 2.6±0.2 MPa, and an elastic modulus of 340±30 MPa, approaching the reported values for cancellous bone. Reverse transcription-polymerase chain reaction showed that HUVEC+HOB coculture on CPC had much higher vascular endothelial growth factor (VEGF) and collagen I expressions than monoculture (p<0.05). Osteogenic markers alkaline phosphatase, osteocalcin (OC), and runt-related transcription factor 2 (Runx2) were also highly elevated. Immunostaining of PECAM1 (CD31) showed abundant microcapillary-like structures on CPC in coculture at 42 days, as HUVEC self-assembled into extensive branches and net-like structures. However, no microcapillary was found on CPC in monoculture. In immunohistochemical staining, the neo-vessels were strongly positive for PECAM1, the von Willebrand factor, and collagen I. Scanning electron microscopy revealed microcapillary-like structures mingling with mineral nodules on CPC. Cell-synthesized minerals increased by an order of magnitude from 4 to 42 days. In conclusion, gas-foaming macroporous CPC was fabricated and HUVEC+HOB coculture was performed for prevascularization, yielding microcapillary-like structures on CPC for the first time. The novel macroporous CPC-microvasculature construct is promising for a wide range of orthopedic applications with enhanced angiogenic and osteogenic capabilities.

Simvastatin‐loaded macroporous calcium phosphate cement: Preparation, in vitro characterization, and evaluation of in vivo performance

The aim of our study was to construct macroporous calcium phosphate bone cements (CPCs) with enhanced osteogenic potential. For this purpose, 300 mM sodium dodecyl sulfate (SDS) as an air-entraining agent was added to the liquid phase and 1, 5, and 10% simvastatin (SIM) was homogenized with the solid phase. The physical and mechanical characteristics of the test samples were investigated. Biological properties of the new CPCs were examined after intramuscular and endosteal implantation in rabbits. The introduction of SDS produced interconnected macropores and did not significantly affect initial setting time, transformation of solid phase to hydroxyapatite, and biocompatibility of CPCs. Large amounts (10 wt %) of SIM could decrease the compressive strength and induce severe muscular necrosis and inflammatory reaction. Small amounts (1 wt %) of SIM were compatible with the CPCs did not affect the physico-chemical properties or biocompatibility and were sufficient to enhance the osteogenic potential of macroporous CPCs.

Basic Properties of Calcium Phosphate Cement Scaffold

Calcium phosphate cement (CPC) sets in situ to form hydroxyapatite and is highly promising for a wide range of clinical applications. However, its low strength limits its use to only non-stress applications, and its lack of macroporosity hinders cell infiltration, bone ingrowth and implant fixation. The aim of this study was to develop strong and macroporous CPC scaffolds by incorporating chitosan and water-soluble mannitol. The incorporation of chitosan could improve the handling properties of CPC. Mannitol provided the needed early strength of CPC and then dissolved to create macropores for tissue ingrowth. This study investigated the effects of mannitol volume fraction (0-70%) on CPC composite mechanical properties and macroporosity of the scaffold after mannitol dissolution.

Human embryonic stem cell encapsulation in alginate microbeads in macroporous calcium phosphate cement for bone tissue engineering

Human embryonic stem cells (hESC) are promising for use in regenerative medicine applications because of their strong proliferative ability and multilineage differentiation capability. To date there have been no reports on hESC seeding with calcium phosphate cement (CPC). The objective of this study was to investigate hESC-derived mesenchymal stem cell (hESCd-MSC) encapsulation in hydrogel microbeads in macroporous CPC for bone tissue engineering. hESC were cultured to form embryoid bodies (EB), and the MSC were then migrated out of the EB. hESCd-MSC had surface markers characteristic of MSC, with positive alkaline phosphatase (ALP) staining when cultured in osteogenic medium. hESCd-MSC were encapsulated in alginate at a density of 1millioncellsml−1, with an average microbead size of 207μm. CPC contained mannitol porogen to create a porosity of 64% and 218-μm macropores, with 20% absorbable fibers for additional porosity when the fibers degrade. hESCd-MSC encapsulated in microbeads in CPC had good viability from 1 to 21days. ALP gene expression at 21days was 25-fold that at 1day. Osteocalcin (OC) at 21days was two orders of magnitude of that at 1day. ALP activity in colorimetric p-nitrophenyl phosphate assay at 21days was fivefold that at 1day. Mineral synthesis by the encapsulated hESCd-MSC at 21days was sevenfold that at 1day. Potential benefits of the CPC-stem cell paste include injectability, intimate adaptation to complex-shaped bone defects, ease in contouring to achieve esthetics in maxillofacial repairs, and in situ setting ability. In conclusion, hESCd-MSC were encapsulated in alginate microbeads in macroporous CPC, showing good cell viability, osteogenic differentiation and mineral synthesis for the first time. The hESCd-MSC-encapsulating macroporous CPC construct is promising for bone regeneration in a wide range of orthopedic and maxillofacial applications.

Periodontal regeneration using an injectable bone cement combined with BMP-2 or FGF-2

Periodontitis is a frequently diagnosed oral disease characterized by bone resorption and soft tissue loss around teeth. Unfortunately, currently available therapies only slow or arrest progress of the disease. Ideally, treatment of periodontal defects should be focused on complete regeneration of the lost tissues [(bone and periodontal ligament (PDL)]. As a result, this study used intrabony defects to evaluate the regenerative potential of an injectable macroporous calcium phosphate cement (CaP) in combination with bone morphogenetic protein-2 (BMP-2) or fibroblast growth factor-2 (FGF-2). After creating 30 periodontal defects in 15 Wistar rats, three treatment strategies were conducted: application of CaP only, CaP + BMP-2 and CaP + FGF-2. Animals were euthanized after 12 weeks and processed for histology and histomorphometry. Using CaP alone resulted in limited effects on PDL and bone healing. CaP + BMP-2 showed a good response for bone healing; a significant 2.4 fold increase in bone healing score was observed compared to CaP. However, for PDL healing, CaP + BMP-2 treatment showed no difference compared to the CaP group. The best results were observed with the combined treatment of CaP + FGF-2, which showed a significant 3.3 fold increase in PDL healing score compared to CaP + BMP-2 and a significant 2.6 fold increase compared to CaP. For bone healing, CaP + FGF-2 showed a significant 1.9 fold increase compared to CaP but no significant difference was noted compared to the CaP + BMP-2 group. The combination of a topical application of FGF-2 and an injectable CaP seems to be a promising treatment modality for periodontal regeneration.

Umbilical cord stem cells released from alginate–fibrin microbeads inside macroporous and biofunctionalized calcium phosphate cement for bone regeneration

The need for bone repair has increased as the population ages. The objectives of this study were to (1) develop a novel biofunctionalized and macroporous calcium phosphate cement (CPC) containing alginate–fibrin microbeads encapsulating human umbilical cord mesenchymal stem cells (hUCMSC) and, for the first time, (2) investigate hUCMSC proliferation and osteogenic differentiation inside the CPC. A macroporous CPC was developed using calcium phosphate powder, chitosan, and a gas-foaming porogen. Five types of CPC were fabricated: a CPC control, CPC+0.05% fibronectin (Fn), CPC+0.1% Fn, CPC+0.1% arginine–glycine–aspartate (RGD), and CPC+0.1% Fn+0.1% RGD. Alginate–fibrin microbeads containing 106hUCMSC per ml were encapsulated in the CPC paste. After the CPC had set, the degradable microbeads released hUCMSC within it. The hUCMSC proliferated inside the CPC, with the cell density after 21days being 4-fold that on day1. CPC+0.1% RGD had the highest cell density, which was 4-fold that of the CPC control. The released cells differentiated along the osteogenic lineage and synthesized bone mineral. The hUCMSC inside the CPC+0.1% RGD construct expressed the genes alkaline phosphatase, osteocalcin and collagen I, at twice the level of the CPC control. Mineral synthesis by hUCMSC inside the CPC+0.1% RGD construct was 2-fold that in the CPC control. RGD and Fn incorporation in the CPC did not compromise its strength, which matched the reported strength of cancellous bone. In conclusion, degradable microbeads released hUCMSC which proliferated, differentiated and synthesized minerals inside the macroporous CPC. The CPC with RGD greatly enhanced cell function. The novel biofunctionalized and macroporous CPC–microbead–hUCMSC construct is promising for bone tissue engineering applications.