Jeong-Hui Park

Direct deposited porous scaffolds of calcium phosphate cement with alginate for drug delivery and bone tissue engineering

This study reports the preparation of novel porous scaffolds of calcium phosphate cement (CPC) combined with alginate, and their potential usefulness as a three-dimensional (3-D) matrix for drug delivery and tissue engineering of bone. An α-tricalcium phosphate-based powder was mixed with sodium alginate solution and then directly injected into a fibrous structure in a Ca-containing bath. A rapid hardening reaction of the alginate with Ca(2+) helps to shape the composite into a fibrous form with diameters of hundreds of micrometers, and subsequent pressing in a mold allows the formation of 3-D porous scaffolds with different porosity levels. After transformation of the CPC into a calcium-deficient hydroxyapatite phase in simulated biological fluid the scaffold was shown to retain its mechanical stability. During the process biological proteins, such as bovine serum albumin and lysozyme, used as model proteins, were observed to be effectively loaded onto and released from the scaffolds for up to more than a month, proving the efficacy of the scaffolds as a drug delivering matrix. Mesenchymal stem cells (MSCs) were isolated from rat bone marrow and then cultured on the CPC-alginate porous scaffolds to investigate the ability to support proliferation of cells and their subsequent differentiation along the osteogenic lineage. It was shown that MSCs increasingly actively populated and also permeated into the porous network with time of culture. In particular, cells cultured within a scaffold with a relatively high porosity level showed favorable proliferation and osteogenic differentiation. An in vivo pilot study of the CPC-alginate porous scaffolds after implantation into the rat calvarium for 6 weeks revealed the formation of new bone tissue within the scaffold, closing the defect almost completely. Based on these results, the newly developed CPC-alginate porous scaffolds could be potentially useful as a 3-D matrix for drug delivery and tissue engineering of bone.

Functional composite nanofibers of poly(lactide-co-caprolactone) containing gelatin-apatite bone mimetic precipitate for bone regeneration

Functional nanofibrous materials composed of gelatin-apatite-poly(lactide-co-caprolactone) (PLCL) were produced using an electrospinning process. A gelatin-apatite precipitate, which mimicked bone extracellular matrix, was homogenized in an organic solvent using various concentrations of PLCL. A fibrous structure with approximate diameters of a few hundred nanometers was successfully generated. Apatite nanocrystallines were found to be effectively distributed within the polymeric matrix of the gelatin-PLCL. The addition of a small amount of gelatin-apatite into PLCL significantly improved the tensile strength of the nanofiber by a factor of 1.8. Moreover, tissue cell growth on the composite nanofiber was enhanced. Osteogenic differentiation of the cells was significantly stimulated by the composite nanofiber compared with the pure PLCL nanofiber. When implanted in a rat calvarium for 6weeks the composite nanofiber supported defect closure and new bone formation better than the pure PLCL nanofiber, as deduced from micro-computed tomography and histological analyses. Based on these results, the gelatin-apatite-PLCL composite nanofiber developed in this study is considered to be potentially useful as a bone tissue regeneration matrix.

Effect of Calcium Phosphate Cements on Growth and Odontoblastic Differentiation in Human Dental Pulp Cells

OBJECTIVE Calcium phosphate cements (CPCs) are an interesting class of bone substitute materials. However, the biological effects of CPCs have not been well studied in human dental pulp cells (HDPCs). The purpose of this study was to investigate the effects of CPCs on the mechanical properties, growth, and odontoblastic differentiation in HDPCs compared with Portland cement (PC) and mineral trioxide aggregate (MTA). METHODS Experimental CPCs either containing chitosan (Ch-CPC) or without chitosan (CPC) were composed from the alpha-tricalcium phosphate powder. Setting time, compressive strength measurements, cell growth, alkaline phosphatase (ALP) activity, the levels of messenger RNA for differentiation-related genes, and mineralization of the HDPCs on various cements were assessed. RESULTS The setting time for CPC-Ch was 7.5 minutes, which was significantly less than the 8.6 minutes for the CPC. On the seventh day of immersion, the compressive strength of CPC-CH reached 13.1 MPa, which was higher than 10.8 MPa of CPC. CPC and Ch-CPC-treated cells showed decreased cell proliferation but increased the levels of ALP activity, enhanced mineralized nodule formation, and upregulated odontoblastic markers messenger RNA including osteonectin, osteopontin, bone sialoprotein, dentin matrix protein-1, matrix extracellular phosphoglycoprotein, and dentin sialophosphoprotein (DSPP), compared with untreated control. The response of CPC and CP-CPC were similar to that of PC and MTA. However, the adhesion, growth, and differentiation in Ch-CPC-treated cells were similar to that in the CPC. CONCLUSION CPC may be useful for pulp-capping applications based on its abilities to promote HDPC differentiation.

Therapeutic bioactive microcarriers: co-delivery of growth factors and stem cells for bone tissue engineering.

Novel microcarriers made of sol-gel-derived bioactive glasses were developed for delivering therapeutic molecules effectively while cultivating stem cells for bone tissue engineering. Silica sols with varying concentration of Ca (0-30 mol.%) were formulated into microspheres ranging from 200 to 300 μm under optimized conditions. A highly mesoporous structure was created, with mesopore sizes of 2.5-6.3 nm and specific surface areas of 420-710 m(2)g(-1), which was highly dependent on the Ca concentration. Therapeutic molecules could be effectively loaded within the mesoporous microcarriers during microsphere formulation. Cytochrome C (cyt C), used as a model protein for the release study, was released in a highly sustainable manner, with an almost zero-order kinetics over a period of months; the amount released was ~2% at 9 days, and 15% at 40 days. A slight increase in the release rate was observed in the microcarrier containing Ca, which was related to the dissolution rate and pore size. The presence of Ca accelerated the formation of hydroxyapatite on the surface of the microcarriers. Cells cultured on the bioactive microcarriers were well adhered and distributed, and proliferated actively, confirming the three-dimensional substrate role of the microcarriers. An in vivo study performed in a rat subcutaneous model demonstrated the satisfactory biocompatibility of the prepared microspheres. As a therapeutic target molecule, basic fibroblast growth factor (bFGF) was incorporated into the microcarriers. A slow release pattern similar to that of cyt C was observed for bFGF. Cells adhered and proliferated to significantly higher levels on the bFGF-loaded microcarriers, demonstrating the effective role of bFGF in cell proliferative potential. It is believed that the developed mesoporous bioactive glass microspheres represent a new class of therapeutic cell delivery carrier, potentially useful in the sustainable delivery of therapeutic molecules such as growth factors, as well as in the support of stem cell proliferation and osteogenesis for bone tissue engineering.

Angiogenesis in bone regeneration: tailored calcium release in hybrid fibrous scaffolds

In bone regeneration, silicon-based calcium phosphate glasses (Bioglasses) have been widely used since the 1970s. However, they dissolve very slowly because of their high amount of Si (SiO2 > 45%). Recently, our group has found that calcium ions released by the degradation of glasses in which the job of silicon is done by just 5% of TiO2 are effective angiogenic promoters, because of their stimulation of a cell-membrane calcium sensing receptor (CaSR). Based on this, other focused tests on angiogenesis have found that Bioglasses also have the potential to be angiogenic promoters even with high contents of silicon (80%); however, their slow degradation is still a problem, as the levels of silicon cannot be decreased any lower than 45%. In this work, we propose a new generation of hybrid organically modified glasses, ormoglasses, that enable the levels of silicon to be reduced, therefore speeding up the degradation process. Using electrospinning as a faithful way to mimic the extracellular matrix (ECM), we successfully produced hybrid fibrous mats with three different contents of Si (40, 52, and 70%), and thus three different calcium ion release rates, using an ormoglass-polycaprolactone blend approach. These mats offered a good platform to evaluate different calcium release rates as osteogenic promoters in an in vivo subcutaneous environment. Complementary data were collected to complement Ca(2+) release analysis, such as stiffness evaluation by AFM, ζ-potential, morphology evaluation by FESEM, proliferation and differentiation analysis, as well as in vivo subcutaneous implantations. Material and biological characterization suggested that compositions of organic/inorganic hybrid materials with a Si content equivalent to 40%, which were also those that released more calcium, were osteogenic. They also showed a greater ability to form blood vessels. These results suggest that Si-based ormoglasses can be considered an efficient tool for calcium release modulation, which could play a key role in the angiogenic promoting process.

Titanium phosphate glass microspheres for bone tissue engineering

We have demonstrated the successful production of titanium phosphate glass microspheres in the size range of ∼10-200 μm using an inexpensive, efficient, easily scalable process and assessed their use in bone tissue engineering applications. Glasses of the following compositions were prepared by melt-quench techniques: 0.5P₂O₅-0.4CaO-(0.1-x)Na₂O-xTiO₂, where x=0.03, 0.05 and 0.07 mol fraction (denoted as Ti3, Ti5 and Ti7 respectively). Several characterization studies such as differential thermal analysis, degradation (performed using a novel time lapse imaging technique) and pH and ion release measurements revealed significant densification of the glass structure with increased incorporation of TiO₂ in the glass from 3 to 5 mol.%, although further TiO₂ incorporation up to 7 mol.% did not affect the glass structure to the same extent. Cell culture studies performed using MG63 cells over a 7-day period clearly showed the ability of the microspheres to provide a stable surface for cell attachment, growth and proliferation. Taken together, the results confirm that 5 mol.% TiO₂ glass microspheres, on account of their relative ease of preparation and favourable biocompatibility, are worthy candidates for use as substrate materials in bone tissue engineering applications.

Biointerface control of electrospun fiber scaffolds for bone regeneration: Engineered protein link to mineralized surface

Control over the interface of biomaterials that favors the initial adhesion and subsequent differentiation of stem cells is one of the key strategies in bone tissue engineering. Here we engineer the interface of biopolymer electrospun fiber matrices with a fusion protein of fibronectin 9-10 domain (FNIII9-10) and osteocalcin (OCN), aiming to stimulate mesenchymal stem cell (MSC) functions, including initial adhesion, growth and osteogenic differentiation. In particular, a specific tethering of FNIII9-10-OCN protein was facilitated by the hydroxyapatite (HA) mineralization of the biopolymer surface through a molecular recognition of OCN to the HA crystal lattice. The FNIII9-10-OCN anchorage to the HA-mineralized fiber was observed to be highly specific and tightly bound to preserve stability over a long period. Initial cell adhesion levels, as well as the spreading shape and process, of MSCs within 24h were strikingly different between the fibers linked with and without fusion protein. Significant up-regulations in the mRNA expression of adhesion signaling molecules occurred with the fusion protein link, as analyzed by the reverse transcriptase polymerase chain reaction. The expression of a series of osteogenic-related genes at later stages, over 2-3weeks, was significantly improved in the fusion protein-tailored fiber, and the osteogenic protein levels were highly stimulated, as confirmed by immunofluorescence imaging and fluorescence-activated cell sorting analyses. In vivo study in a rat calvarium model confirmed a higher quantity of new bone formation in the fiber linked with fusion protein, and a further increase was noticed when the MSCs were tissue-engineered with the fusion protein-linked fiber. Collectively, these results indicate that FN-OCN fusion protein links via HA mineralization is a facile tool to generate a biointerface with cell-attractive and osteogenic potential, and that the engineered fibrous matrix is a potential bone regenerative scaffold.

Silk fibroin–polyurethane blends: Physical properties and effect of silk fibroin content on viscoelasticity, biocompatibility and myoblast differentiation

As a way to modify both the physical and biological properties of a highly elastic and degradable polyurethane (PU), silk fibroin (SF) was blended with the PU at differing ratios. With increasing SF content, the tensile strength decreased as did the strain at break; the stiffness increased to around 35 MPa for the highest silk content. C2C12 (a mouse myoblast cell line) cells were used for in vitro experiments and showed significantly improved cell responses with increasing SF content. With increasing SF content the number of non-adherent cells was reduced at both 4 and 8h compared to the sample with the lowest SF content. In addition, muscle marker genes were upregulated compared to the sample containing no SF, and in particular sarcomeric actin and α-actin.

Mineralized poly(lactic acid) scaffolds loading vascular endothelial growth factor and the in vivo performance in rat subcutaneous model

The functionalization of degradable polymeric scaffolds with therapeutic molecules such as vascular endothelial growth factor (VEGF) is a key strategy to gain better regenerative ability of damaged bone tissue by stimulating vascularization and tissue perfusion. Here, we combined VEGF with poly(lactic acid) (PLA) porous scaffold, after modifying the PLA surface with calcium phosphate (CaP) mineral. The mineralized PLA scaffold (mPLA) showed more effective loading capacity of VEGF than the PLA without mineralization as well as profiled sustainable release of VEGF for up to a couple of weeks. The VEGF-loaded mPLA scaffold presented significantly improved proliferation of primary endothelial cells for up to 7 days, with respect to the scaffold without the VEGF loading. The performance of the engineered scaffold was assessed after subcutaneous implantation in rats for 4 weeks. Histological results showed favorable tissue compatibility of both the mPLA scaffolds (with and without VEGF loading), as characterized by infiltration of inflammatory cells, formation of fibrous capsule, and ingrowth of fibroblasts into the matrices. Immunohistochemical staining of the von Willebrand Factor revealed significantly improved formation of neo-capillaries in the VEGF-loaded mPLA. Based on this study, the strategy of VEGF loading onto mineralized PLA scaffold is considered beneficial for gaining improved vascularization of the polymeric scaffolds, suggesting potential applications for bone tissue engineering.

Tethering bi-functional protein onto mineralized polymer scaffolds to regulate mesenchymal stem cell behaviors for bone regeneration

Modifying three-dimensional scaffolds with bioactive extracellular matrix (ECM) molecules enhances their potential use in tissue engineering, by providing natural biochemical/physical cues for cell recognition. Here, we engineered the surface of poly(caprolactone) (PCL) scaffolds, first with bone mineral hydroxyapatite (HA), and then with fibronectin-osteocalcin (FN-OCN) bi-functional protein by means of affinity binding between OCN and HA. While FN is expected to enhance initial adhesion of immature precursor cells, OCN is considered to regulate osteogenic differentiation. Quartz crystal microbalance dissipation analysis revealed FN-OCN protein had a more stable and stronger adherence to the HA-mineralized surface than to the native PCL-surface. Initial adhesion and the spreading of rat mesenchymal stem cells were significantly enhanced on the FN-OCN tethered scaffold. Expression of bone-associated genes (osteopontin, bone sialoprotein II and OCN) was significantly higher on the FN-OCN tethered scaffold. Moreover, those proteins were more abundantly found when cultured on the scaffolds with FN-OCN than those without, as confirmed by immunofluorescence cell labeling and fluorescence activated cell sorting analysis. All taken, the tethering of FN-OCN to a HA-mineralized surface is an effective strategy to provide biopolymer scaffolds improved bi-functional capacity for bone tissue engineering, in terms of initial cell adhesion and osteogenic differentiation.