Bioactive Nanocomposite Scaffolds by Melt Electrospinning of Poly-ε-Caprolactone/Polyethylene Oxide/Polyethylene Glycol-Hydroxyapatite Blends for Bone Tissue Engineering.

The aim of this study was to evaluate the effects of maxillary sinus floor elevation by a tissue-engineered bone complex with recombinant human bone morphogenetic protein-2 (rhBMP-2)-loaded porous calcium phosphate cement (CPC) scaffold and bone marrow stromal cells (bMSCs) in rabbits. bMSCs were cultured and osteogenically induced. The osteoblastic differentiation of expanded bMSCs was detected by alkaline phosphatase activity, and calcium deposits in vitro. Thirty-six rabbits were randomly allocated into week 2, 4 and 8 observation groups. At each time point, 24 maxillary sinus floor elevation surgeries in 12 rabbits were performed bilaterally and randomly implanted by (1) CPC materials alone (group A, n = 6), (2) rhBMP-2/CPC composite materials alone (group B, n = 6), (3) CPC/bMSCs complex (group C, n = 6) and (4) rhBMP-2/CPC/bMSCs complex (group D, n = 6). As for maxillary sinus floor elevation, rhBMP-2-loaded CPC could promote new bone formation as compared to CPC, while addition of bMSCs could further enhance its new bone formation and maturity significantly, as detected by histological findings, and fluorochrome labeling. Our data suggested that rhBMP-2/CPC possessed excellent osteoinductive ability, while combining with bMSCs could further promote new bone formation and maturation in maxillary sinus elevation.

Calcium phosphate cement (CPC) sets in situ to form solid hydroxyapatite, can conform to complex cavity shapes without machining, has excellent osteoinductivity, and is able to be resorbed and replaced by new bone. Therefore, CPC is promising for craniofacial and orthopaedic repairs. However, its low strength and lack of macroporosity limit its use. This study investigated CPC reinforcement with absorbable fibers, the effects of fiber volume fraction on mechanical properties and macroporosity, and the biocompatibility of CPC-fiber composite. The liquid phase of CPC in this study was the weak acidic solution of chitosan. Chitosan has favourable biocompatibility, which has high viscosity in solution. The incorporation of chitosan could improve the handling properties of CPC. The liquid phase contained citric acid could strongly improve the hydration rate of CPC, which shortened the setting time and increased the compressive strength of CPC. In addition, the weak acidic environment around the biomaterials could accelerate the degradation of CPC, which was important to bone tissue engineering. The rationale was that large-diameter absorbable fibers would initially strengthen the CPC graft, then dissolve to form long cylindrical macropores for colonization by osteoblasts. Compressive strength was measured vs. fiber volume fraction from 0% (CPC Control without fibers) to 70%. Animal experiment showed that the material had osteoinductivity and biodegradability when the material was implanted into the muscle pouches in the thigh of rabbits. Compressive strength (mean ± SD; n=3) of CPC with 70% fibers was 0.8± 0.1 MPa. Long cylindrical macropores 100~300 μm in diameter were created in CPC after fiber dissolution, and the CPC-fiber scaffold reached a total porosity of 75.1±1.2% with 70% fibers. The new CPC-fiber formulation had good potentiality of ectopic bone induction. The method of using large-diameter absorbable fibers in bone graft for mechanical properties and formation of long cylindrical macropores for bone ingrowth may be applicable to other tissue engineering materials.

Effects of electrospun submicron fibers in calcium phosphate cement scaffold on mechanical properties and osteogenic differentiation of umbilical cord stem cells

Objective To investigate the cell toxicity of a novel macropores calcium phosphate cement (CPC) scaffold and its influence on cell adhesion,growth and proliferation.Methods A novel CPC material was synthesized by means of adding mannitol porogens and applying sodium solution as the cement liquid.The cell growth and proliferation in the novel CPC material extraction was observed by CCK8 assay.Scanning electron microscopy was used to observe hole diameter of the material,cell adhesion and growth in the material.The experiment of three point bending was used to test the biomechanic performance of the CPC material.Results The novel CPC material reached hole diameter value of (267.43±118.01)μm,microporosity of (66.15±6.91)％.Maximum load,flexural strength and toughness of the novel CPC material was increased about one time compared to the traditional CPC (P＜0.05).CCK8 assay showed there were no significant difference of the light absorption value of cells in the CPC extraction in the 4th,6th,8th day compare to the control group (P＞0.05).Conclusion The novel CPC material has the strong biomechanics performance,macropores,high microporosity and excellent biocompatibility,which is promising for ideal bone tissue engineering scaffold. Key words: Calcium phosphate cement; Mannitol; Bone tissue engineering; Biomechanics; Biocompatibility

The objective of this study was to assess the effects of maxillary sinus floor elevation with a tissue-engineered bone constructed with bone marrow stromal cells (bMSCs) and calcium-magnesium phosphate cement (CMPC) material. The calcium (Ca), magnesium (Mg), and phosphorus (P) ions released from calcium phosphate cement (CPC), magnesium phosphate cement (MPC), and CMPC were detected by inductively coupled plasma atomic emission spectroscopy (ICP-AES), and the proliferation and osteogenic differentiation of bMSCs seeded on CPC, MPC, and CMPC or cultured in CPC, MPC, and CMPC extracts were measured by MTT analysis, alkaline phosphatase (ALP) activity assay, alizarin red mineralization assay, and real-time PCR analysis of the osteogenic genes ALP and osteocalcin (OCN). Finally, bMSCs were combined with CPC, MPC, and CMPC and used for maxillary sinus floor elevation in rabbits, while CPC, MPC, or CMPC without cells served as control groups. The new bone formation in each group was detected by histological finding and fluorochrome labeling at weeks 2 and 8 after surgical operation. It was observed that the Ca ion concentrations of the CMPC and CPC scaffolds was significantly higher than that of the MPC scaffold, while the Mg ions concentration of CMPC and MPC was significantly higher than that of CPC. The bMSCs seeded on CMPC and MPC or cultured in their extracts proliferated more quickly than the cells seeded on CPC or cultured in its extract, respectively. The osteogenic differentiation of bMSCs seeded on CMPC and CPC or cultured in the corresponding extracts was significantly enhanced compared to that of bMSCs seeded on MPC or cultured in its extract; however, there was no significant difference between CMPC and CPC. As for maxillary sinus floor elevation in vivo, CMPC could promote more new bone formation and mineralization compared to CPC and MPC, while the addition of bMSCs could further enhance its new bone formation ability significantly. Our data suggest that CMPC possesses moderate biodegradability and excellent osteoconductivity, which may be attributed to its Ca and Mg ion composition, and the tissue-engineered bone constructed of CMPC and bMSCs might be a potential alterative graft for maxillofacial bone regeneration.

Bone substitute materials such as calcium phosphate cements (CPC) are frequently used as growth factor carriers for the stimulation of osteoblast-formation around an implant. However, biological modification based on delicate protein factors like extracellular matrix proteins or growth factors is subject to a number of shortcomings like the need for storage below room temperature and cost of production. The aim of this study was to investigate ionic modification as an alternative bioinorganic route for implant modification. Although it is known that Cu(II) plays a role in angiogenesis and bone formation, not all involved processes are well understood yet. In this study the in vitro effect of Cu(II) on growth and activity of osteoblastic cells seeded on brushite (CaHPO(4) · 2 H(2) O) scaffolds as well as on glass discs was investigated. The results show that Cu(II) enhances cell activity and proliferation of osteoblastic cells on CPC and furthermore affects the expression of several bone specific proteins such as bone sialo protein or osteocalcin. Therefore, the modification of CPC with Cu(II) may offer a promising alternative to protein based modification to stimulate cellular activity for an improved bone healing.

In this research, new bioactive nanocomposite scaffolds were successfully developed using poly(ε-caprolactone) (PCL), cross-linked gelatin and nanoparticles of hydroxyapatite (HAp) after testing different solvents and methods. First, HAp powder was synthesized via a chemical precipitation technique and characterized. Then, the nanocomposites were prepared through layer solvent casting combined with freeze-drying and lamination techniques. According to the results, the increasing of the PCL weight in the scaffolds led to the improvement of the mechanical properties. The amount of ultimate stress, stiffness and also elastic modulus increased from 8 MPa for 0% wt PCL to 23.5 MPa for 50% wt PCL. The biomineralization study revealed the formation of an apatite layer on the scaffolds after immersion in simulated body fluid (SBF). The Ca-P ratios were in accordance to nonstoichiometric biological apatite, which was approximately 1.67. The in vitro biocompatibility and cytocompatibility of the scaffolds were tested using mesenchymal stem cells (MSCs), and the results indicated no sign of toxicity, and cells were found to be attached to the scaffold walls. The in vivo biocompatibility and osteogenesis of these scaffolds in the animal experiments is also under investigation, and the result will be published at the end of the study.

Growth stimulation of periimplant tissues by growth factors like transforming growth factor-beta1 (TGF-beta1) may increase the indication for and success of implant use. Calcium phosphate as a material for implants or for coating of implants is known for its good biologic interaction with bone. Therefore, calcium phosphate implants combined with TGF-beta1 might improve osseointegration. In this study we hypothesise that the addition of recombinant human TGF-beta1 (rhTGF-beta1) to calcium phosphate cement (CPC) affects the differentiation of bone cells growing on the cement layer. rhTGF-beta1 incorporated during setting in a CPC layer at 20 ng rhTGF-beta1/60 mg cement was found to be gradually released into tissue culturing medium leading to a 20% release after 24 h. Two cell populations were obtained from collagenase-treated fragments of adult rat long bones: preosteoblastic cells, which were released by the collagenase treatment, and osteoblastic cells, which grew from the collagenase-stripped bone fragments. Both cell populations were tested for their osteoblastic characteristic phenotype by measuring their alkaline phosphatase (ALP) activity after vitamin D treatment and cyclic AMP after parathyroid hormone stimulation. After preculture the cells were plated on a layer of CPC containing 0 (control), 10, or 20 ng rhTGF-beta1/60 mg CPC. Bone cell differentiation was analyzed after 10 days by measuring the ALP activity, as well as the protein content of the cell layer. Incorporation of rhTGF-beta1 in the CPC did not change the ALP activity in osteoblastic cells, but a significant (analyzed by multivariate analysis of variance) increase was observed in preosteoblastic cells. Incorporation of 10 ng of rhTGF-beta1 in 60 mg of CPC increased the ALP activity in preosteoblastic cells by threefold and 20 ng rhTGF-beta1/60 mg CPC increased it by fivefold. The total protein content was not affected by rhTGF-beta1 in either of the cell populations. We conclude that rhTGF-beta1 incorporated during setting in CPC stimulates the differentiation of preosteoblastic cells in vitro. These results provide a basis for further studies on the use of this combination as an implant material in vivo.

Calcium phosphate cement (CPC) has been widely used in bone tissue repairing due to its physical mechanical properties and biocompatibility. Addition of trace element to CPC has shown promising evidence to improve the physical properties and biological activities of CPC. Lithium (Li) has effect on osteoblast proliferation and differentiation. In this study, we incorporated Li to CPC and examined the physical properties of Li/CPC and its effect on osteoblast proliferation and differentiation. We found that Li doped CPC maintained similar setting time, pore size distribution, compressive strength, composition, and morphology as CPC without Li. Additionally, Li doped CPC improved osteoblast proliferation and differentiation significantly compared to CPC without Li. To our knowledge, our results, for the first time, show that Li doped CPC has beneficial effect on osteoblast in cell culture while keeps the excellent physical-mechanical properties of CPC. This study will lead to potential application of Li doped CPC in bone tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 944-952, 2017.

Self-hardened calcium phosphate cement (CPC) sets to form hydroxyapatite and possesses excellent osteoconductivity. However, lack of macroporosity and low strength constrain its application in bone tissue engineering. Recent studies have incorporated various fibres into CPC to improve its mechanical strength. The present approach focused on the reinforcement of CPC with chitosan fibres and then the effects of the fibre structure on the mechanical properties and macrochannels formation characteristics of CPC-fibre composite were investigated. Chitosan fibres of diameter 200 microm were used to fabricate two types of three-dimensional structure, which were then coated with collagen and incorporated into CPC to fabricate CPC-fibre implants with a fibre volume content of 5 per cent. The compressive strength of the CPC-fibre implant was 33 MPa when the strain was 2.4 per cent, which is fourfold higher than that of the CPC control. Nine cylindrical implants including six CPC-fibre implants were implanted in the bone defects of nine dogs and were then post-operatively observed. After 20 weeks in vivo, new callus from the healthy tissue of the defect entirely integrated with the CPC-fibre implant and new bone was formed as the implant degraded. Scanning electronic microscopy images indicated that macrochannels were formed in the CPC-fibre implants with the degradation of fibres, but only micropores with a scale of less than 50 microm could be observed in the CPC control. Briefly, the incorporation of a suitable chitosan-fibre structure into a CPC implant not only could improve its mechanical properties but also facilitated the bone repair process in vivo.

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

One of the goals of tissue engineering is to create composites that can mimic the behavior of bone in terms of various properties and lead to faster healing with minimal side effects. In this regard, in the present study, porous nanocomposite scaffolds based on Poly(Xylitol-Dodecanedioic acid) (PXDDA) and polycaprolactone (PCL) were prepared in the presence and absence of hydroxyapatite (HA) nanoparticles (NPs) using a salt leaching method with sodium chloride as porogen particles. The results of microscopic examination showed that the presence of HA NPs significantly increased the porosity of the polymer-based scaffold and contact angle (CA) tests showed that these NPs greatly improved the hydrophilicity of the samples. In addition, element mapping and XRD analyses indicated good dispersion of the HA NPs in the polymer matrix. The FTIR results also showed that the sample containing 70% PXDDA and 30% PCL with HA NPs had the highest hydroxyl, hydrocarbon, and ester linkages. In addition, compression tests and dynamic mechanical analysis (DMA) showed that the inclusion of the NPs weakened the mechanical properties of the nanocomposite produced. In addition, the results of the MTT test showed that the presence of HA NPs in this polymer matrix reduced toxicity and increased cell viability.

Calcium phosphate cements (CPCs) have been widely used as bone substitute materials. However, their degradation property is insufficient to stimulate bone healing in large bone defects, besides, the poor initial mechanical strength limits their application to non-load bearing areas. In this study, to overcome the drawbacks of CPCs, magnesium phosphate cements (MPCs) characterized by high initial strength and relatively rapid degradation were combined with CPCs to develop novel calcium-magnesium phosphate cements (CMPCs). The morphology of the CPC sample consisted of needle-like crystals, whereas the interlinked hemispherical globules were observed for the CMPCs. The handling and mechanical characteristics of the samples as well as their degradation behavior under in vitro condition were investigated. Results showed that the CMPCs exhibited shorter setting time and higher compressive strength than the CPC. In addition, CMPCs showed significatnly improved degradability compared to the CPC in Tris-HCl buffer solution.

The vast majority of materials used in bone tissue engineering and regenerative medicine are based on calcium phosphates due to their similarity with the mineral phase of natural bone. Among them, calcium phosphate cements, which are composed of a powder and a liquid that are mixed to obtain a moldable paste, are widely used. These calcium phosphate cement pastes can be injected using minimally invasive surgery and adapt to the shape of the defect, resulting in an entangled network of calcium phosphate crystals. Adding an organic phase to the calcium phosphate cement formulation is a very powerful strategy to enhance some of the properties of these materials. Adding some water-soluble biocompatible polymers in the calcium phosphate cement liquid or powder phase improves physicochemical and mechanical properties, such as injectability, cohesion, and toughness. Moreover, adding specific polymers can enhance the biological response and the resorption rate of the material. The goal of this study is to overview the most relevant advances in this field, focusing on the different types of polymers that have been used to enhance specific calcium phosphate cement properties.

Mineralization and drug release of hydroxyapatite/poly(l-lactic acid) nanocomposite scaffolds prepared by Pickering emulsion templating