In vitro toxicity assessment of crosslinking agents used in hyaluronic acid dermal filler.

Hyaluronic acid (HA) dermal fillers are produced by crosslinking HA with agents, such as 1,4-butanediol diglycidyl ether (BDDE) and poly (ethylene glycol) diglycidyl ether (PEGDE) to acquire desired properties. Thus, the safety evaluation of these crosslinkers is needed at the cellular level. In the present study, cell viability, cytotoxicity, membrane integrity, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and inflammatory responses were evaluated in the human keratinocyte cell line, HaCaT and human dermal fibroblast cell line, HDF in response to treatment with the crosslinkers. In both the cell lines, BDDE significantly decreased cell viability at 100-1000 ppm, while PEGDE showed a decrease at 500-1000 ppm. In HaCaT cells, BDDE markedly increased cytotoxicity (lactate dehydrogenase release) at 100-1000 ppm, but PEGDE showed an increase at 500-1000 ppm. Cells treated with BDDE (100 ppm) caused alteration in the integrity of cell membrane and shape. In both the cell lines, BDDE-treated cells showed significantly higher ROS levels and MMP loss than PEGDE-treated cells. Also, BDDE-treated cells exhibited higher COX-2 expression at 100 ppm. Expression of inflammatory cytokines (TNF-α, and IL-1 ß) was higher in BDDE-treated cells. Taken together, PEGDE-treated cells showed markedly lower cytotoxicity, ROS production, and inflammatory responses than BDDE-treated cells. Our data suggest that PEGDE is safer than BDDE as a crosslinker in HA dermal fillers.

In vivo release of bovine serum albumin from an injectable small intestinal submucosa gel.

We aimed to develop a delivery system capable of maintaining a sustained release of protein drugs at specific sites using potentially biocompatible biomaterials. Here, we used bovine serum albumin (BSA) as a test protein to explore the potential utility of an injectable small intestine submucosa (SIS) as a depot for protein drugs. The prepared SIS powder was dispersed in PBS. The SIS suspension easily entrapped BSA in pharmaceutical formulations at room temperature. When this was suspension subcutaneously injected into rats, it gelled, forming an interconnecting three-dimensional network SIS structure to allow BSA to penetrate through it. The amount of BSA-FITC released from the SIS gel was determined in rat plasma and monitored by real-time in vivo molecular imaging. The data indicated the sustained release of BSA-FITC for 30 days in vivo. In addition, SIS gel provoked little inflammatory response. Collectively, our results show that the SIS gel described here could serve as a minimally invasive therapeutics depot with numerous benefits compared to other injectable biomaterials.

Stem cell response to multiwalled carbon nanotube-incorporated regenerated silk fibroin films.

Multiwalled carbon nanotubes (MWCNTs) are considered to be the ideal reinforcements for biorelated applications on account of their remarkable structural, mechanical and thermal properties. However, before MWCNTs can be incorporated into new and existing biomedical devices, their toxicity and biocompatibility need to be investigated thoroughly. In this study, regenerated silk fibroin/MWCNT nanocomposite films were prepared using a solvent system with pre-dispersed MWCNTs. Their biocompatibility was examined in vitro using human bone marrow stem cells. Scanning electron microscopy and a WST-1 assay demonstrated that the silk fibroin/MWCN film supported BMSC attachment and growth over 7 days in culture similar to the silk fibroin only film.

Potential induction of rat muscle-derived stem cells to neural-like cells by retinoic acid.

Several recent studies have demonstrated that stem cell differentiation can be generated by derivatives of retinoic acid. In this study we chose retinoic acid (RA) for inducing neural differentiation of rat muscle-derived stem cells (rMDSCs). rMDSCs were pre-induced with 10 ng/ml basic fibroblast growth factor (bFGF) and then treated with 2 µM RA. After stimulation, RA induced rMDSCs to have a neural-like morphology after 1-7 days of in vitro differentiation. In the results of immunocytochemistry, rMDSC treated with RA showed abundant positive cells against the neuronal markers neuronal-specific enolase (NSE) and tubulin-ßIII (Tuj1). Also, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase)-positive cells were observed, indicating oligodendrocyte lineage cells. However, positive cells against glial fibrillary acidic protein (GFAP), marker of astrocytes, were not detected. The mRNA profile of these cells included higher expression of NSE compared with those of non-treated cells in real-time PCR. From the data in this work, we suggest that rMDSCs can trans-differentiate into a neural-like phenotype under the RA conditions.

In vivo efficacy of paclitaxel-loaded injectable in situ-forming gel against subcutaneous tumor growth.

Injectable in situ-forming gels have received considerable attention as localized drug delivery systems. Here, we examined a poly(ethylene glycol)-b-polycaprolactone (MPEG-PCL) diblock copolymer gel as an injectable drug depot for paclitaxel (Ptx). The copolymer solution remained liquid at room temperature and rapidly gelled in vivo at body temperature. In vitro experiments showed that Ptx was released from MPEG-PCL copolymer gels over the course of more than 14 days. Experiments employing intratumoral injection of saline (control), gel-only, Taxol, or Ptx-loaded gel into mice bearing B16F10 tumor xenografts showed that Ptx-loaded gel inhibited the growth of B16F10 tumors more effectively than did saline or gel alone. Further, intratumoral injection of Ptx-loaded gel was more efficacious in inhibiting the growth of B16F10 tumor over 10 days than was injection of Taxol. A histological analysis demonstrated an increase in necrotic tissue in tumors treated with Ptx-loaded gel. In conclusion, our data show that intratumoral injection of Ptx-loaded MPEG-PCL diblock copolymer yielded an in situ-forming gel that exhibited controlled Ptx release profile, and that was effective in treating localized solid tumors.

Small intestine submucosa sponge for in vivo support of tissue-engineered bone formation in the presence of rat bone marrow stem cells.

The aim of the current study was to visualize new bone formed in vivo on a small intestine submucosa (SIS) sponge used as a tissue-engineered scaffold for the repair of damaged bone. The SIS sponge provided a three-dimensional pore structure, and supported good attachment and viability of rat bone marrow stem cells (rBMSCs). To examine bone regeneration, we prepared full-thickness bilateral bone defects in the rat crania, and then treated the defects with an implanted SIS sponge or PGA mesh without or with rBMSCs, or left the defects untreated. Bone defects were evaluated by micro-CT and histologically after 2 and 4 weeks. Micro-CT demonstrated a trend toward a decrease in bone void in both the SIS sponge and SIS sponge/rBMSCs groups compared to the control and PGA mesh groups. At 4 weeks, bone formation in defects containing SIS sponge/rBMSCs was significantly greater than in all other groups. A histological analysis after 2 and 4 weeks of implantation showed localized collagen and osteocalcin deposition on SIS sponges and SIS sponges with rBMSCs. These in vivo results indicate that the SIS sponge, implanted at bone-removal defects, facilitated bone regeneration.

Induction of neurogenesis in rat bone marrow mesenchymal stem cells using purine structure-based compounds.

Rat bone marrow stem cells (rBMSCs) in the presence of chemical molecules were differentiated into neurons.

Electrostatic crosslinked in situ-forming in vivo scaffold for rat bone marrow mesenchymal stem cells.

We herein formulated and characterized an in situ-forming gel consisting of sodium carboxymethylcellulose (CMC) and poly(ethyleneimine) (PEI) and examined its use as an in vivo scaffold for rat bone marrow stem cells (rBMSCs). The changes of zeta potential, size, and viscosity of CMC solutions with 0 to 30 wt% PEI confirmed the electrostatic interaction and temperature-dependence between anionic CMC and cationic PEI. The CMC/PEI solution produced an electrostatically crosslinked gel with a three-dimensional network structure. The CMC solution containing 10 wt% PEI transformed to a gel at temperatures greater than 35 degrees C and was chosen for subcutaneous injection into rats. The CMC/PEI (90/10) gel with pore structure acted as a suitable biocompatible substrate for the in vitro and in vivo attachment and proliferation of rBMSCs. As the CMC/PEI (90/10) solution with and without rBMSCs was injected into Fisher rats, it became a gel in the subcutaneous dorsum of the rats and maintained its shape even after 28 days in vivo. The injected rBMSCs survived in the CMC gel for 28 days. Injection of CMC/PEI gel alone induced macrophage accumulation in the host tissue and at the edge of the gel, whereas injection of CMC/PEI gel containing rBMSCs was associated with less macrophage accumulation, indicating immunosuppression by the transplanted rBMSCs. Our results collectively show that CMC/PEI gel could serve as an in situ-forming gel scaffold for entrapped rBMSCs in vivo.

Injectable CMC/PEI gel as an in vivo scaffold for demineralized bone matrix.

A number of materials have been considered as sources of grafts to repair bone defects. Here, we examined the possibility of creating in situ-forming gels from sodium carboxymethylcellulose (CMC) and poly(ethyleneimine) (PEI) for use as an in vivo carrier of demineralized bone matrix (DBM). The interaction between anionic CMC and cationic PEI was examined by evaluating phase transition behavior and viscosity of CMC solutions containing 0-30 wt% PEI. CMC solutions containing 10 wt% PEI exhibited a sol-to-gel phase transition at temperatures greater than 35 degrees C. The phase transition is caused by electrostatic crosslinking of the CMC/PEI solution to form a gel with a three-dimensional network structure. In situ-formed gel implants were successfully fabricated in vivo by simple subcutaneous injection of the CMC/PEI (90/10) solution (with and without DBM) into Fisher rats. The resulting in situ-formed implant maintained its shape for 28 days in vitro and in vivo. Our results show that in situ-forming CMC/PEI gels can serve as a DBM carrier that can be delivered with a minimally invasive procedure.