Da Yeon Kim

In vivo efficacy of an intratumorally injected in situ-forming doxorubicin/poly(ethylene glycol)-b-polycaprolactone diblock copolymer.

The effectiveness of systemically administered anticancer treatments is limited by difficulties in achieving therapeutic doses within tumors, a problem that is complicated by dose-limiting side effects to normal tissue. This work examined injectable in situ-forming gels as a localized drug-delivery system. An MPEG-PCL (MP) solution containing doxorubicin (Dox) existed in an emulsion-sol state at room temperature and rapidly gelled in vitro and in vivo at body temperature. The release of Dox from Dox-loaded MP gels was sustained in vitro over 20 days after an initial burst, indicating that the MP gel acted as a drug depot. Dox-loaded MP gels exhibited remarkable in vitro anti-proliferative activities against B16F10 cancer cells. In vivo experiments employing B16F10 cancer cell xenograft-bearing mice showed that a single intratumoral injection of Dox-loaded MP gel inhibited the growth of tumors as effectively as repeated injections of free Dox, and more effectively than a single dose of free Dox, or saline or gel alone. Consistent with the observed suppression of tumor growth, intratumorally injected free Dox or Dox released from Dox-loaded MP gels caused apoptosis of tumor cells. The tumor biodistribution of free Dox after 1 day was ∼90%, which dropped to ∼15% after 4 days. The biodistribution of Dox following a single injection of Dox-loaded MP gel was also ∼90% on day 1, but remained at ∼13%, even after 15 days. Only a small amount of Dox was found in other organ tissues following intratumoral injection, implying fewer off-target side effects.

Tissue engineered regeneration of completely transected spinal cord using human mesenchymal stem cells

The present study employed a combinatorial strategy using poly(D,L-lactide-co-glycolide) (PLGA) scaffolds seeded with human mesenchymal stem cells (hMSCs) to promote cell survival, differentiation, and neurological function in a completely transected spinal cord injury (SCI) model. The SCI model was prepared by complete removal of a 2-mm length of spinal cord in the eighth-to-ninth spinal vertebra, a procedure that resulted in bilateral hindlimb paralysis. PLGA scaffolds 2 mm in length without hMSCs (control) or with different numbers of hMSCs (1 × 10(5), 2 × 10(4), and 4 × 10(3)) were fitted into the completely transected spinal cord. Rats implanted with hMSCs received Basso-Beattie-Bresnahan scores for hindlimb locomotion of about 5, compared with ~2 for animals in the control group. The amplitude of motor-evoked potentials (MEPs) averaged 200-300 μV in all hMSC-implanted SCR model rats. In contrast, the amplitude of MEPs in control group animals averaged 135 μV at 4 weeks and then declined to 100 μV at 8 weeks. These results demonstrate functional recovery in a completely transected SCI model under conditions that exclude self-recovery. hMSCs were detected at the implanted site 4 and 8 weeks after transplantation, indicating in vivo survival of implanted hMSCs. Immunohistochemical staining revealed differentiation of implanted hMSCs into nerve cells, and immunostained images showed clear evidence for axonal regeneration only in hMSC-seeded PLGA scaffolds. Collectively, our results indicate that hMSC-seeded PLGA scaffolds induced nerve regeneration in a completely transected SCI model, a finding that should have significant implications for the feasibility of therapeutic and clinical hMSC-delivery using three-dimensional scaffolds, especially in the context of complete spinal cord transection.

An injectable biodegradable temperature-responsive gel with an adjustable persistence window

ɛ-Caprolactone (CL) and 3-benzyloxymethyl-6-methyl-1,4-dioxane-2,5-dion (fLA), with a benzyloxymethyl group at the 3-position of the lactide, were randomly copolymerized. The methoxy polyethylene glycol (MPEG)-b-[poly(ɛ-caprolactone)-ran-poly(3-benzyloxymethyl lactide) (PCL-ran-PfLA)] diblock copolymers were designed such that the PfLA content (0-15 mol%) in the PCL segment was varied. The MPEG-b-(PCL-ran-PfLA) diblock copolymers were derivatized by introducing a pendant benzyl group (MC(x)L(y)-OBn), hydroxyl group (MC(x)L(y)-OH), or carboxylic acid group (MC(x)L(y)-COOH) at the PfLA segment. The derivatized MPEG-b-(PCL-ran-PfLA) diblock copolymer solutions exhibited sol-to-gel phase transitions upon a temperature increase. The sol-to-gel phase transition depended on both the type of functional pendant group on the PfLA and the PfLA content in the PCL segment. MC(x)L(y)-COOH diblock copolymer solutions formed gels immediately after injection into Fischer rats. The gels gradually degraded over a period of 0-6 weeks after the initial injection, and the rate of degradation increased for higher concentrations of PfLA. Immunohistochemical characterization showed that the in vivo MPEG-b-(PCL-ran-PfLA) diblock copolymer gels provoked only a modest inflammatory response. These results show that the MPEG-b-(PCL-ran-PfLA) diblock copolymer gel described here may serve as a minimally invasive therapeutic, in situ-forming gel system with an adjustable temperature-responsive and in vivo biodegradable window.

Small intestine submucosa and mesenchymal stem cells composite gel for scarless vocal fold regeneration

The purpose of this study is to demonstrate scarless vocal fold (VF) regeneration by using a composite gel composed of small intestine submucosa (SIS) and mesenchymal stem cells (MSCs). A scar was made with an electrocoagulator on both VFs in 24 rabbits, followed by injection of either MSCs, SIS, or MSCs-SIS composite gel in the right side VF, while the left side VF was left untreated. VF scars were evaluated with in vivo fluorescence live imaging system (IFLIS), endoscopy, histology, and videokymography (VKG) after eight weeks. IFLIS demonstrated that SIS enabled the MSCs to survive and be engrafted in the VF. The histological analysis showed increased hyaluronic acid accumulation and controlled collagen deposition by MSCs-SIS composite gel. VKG analysis showed more favorable vibrations of MSCs-SIS injected VF, compared to other treatment group. In conclusion, the injectable SIS supplied a niche for the MSCs to stably settle down in scarred VFs and helped to regulate ECM synthesis. The ECM remodeling underwent by the surviving MSCs eventually led to the functional improvement of the VF. The results of the present investigation suggest that SIS-MSCs composite gel is a plausible biomaterial for prolonged survival of MSCs in VFs and promotes scarless VF healing.

Injectable intratumoral hydrogel as 5-fluorouracil drug depot

The effectiveness of systemically administered anticancer treatments is limited by difficulties in achieving therapeutic doses within tumors, a problem that is complicated by dose-limiting side effects to normal tissue. To increase the efficacy and reduce the toxicity of systemically administered anticancer 5-fluorouracil (5-Fu) treatments in patients, intratumoral administration of an injectable hydrogel has been evaluated in the current work. The MPEG-b-(PCL-ran-PLLA) diblock copolymer (MCL) containing 5-Fu existed in an emulsion-sol state at room temperature and rapidly gelled in vivo at the body temperature. MCL acted as in vivo biodegradable drug depot over a defined experimental period. A single injection of 5-Fu-loaded MCL solution resulted in significant suppression of tumor growth, compared with repeated injection of free 5-Fu as well as saline and MCL alone. For both repeated injections of free 5-Fu and single injection of 5-Fu-loaded MCL, most of the 5-Fu was found in the tumor, indicating the maintenance of therapeutic concentrations of 5-Fu within the target tumor tissue and the prevention of systemic toxicity associated with 5-Fu in healthy normal tissues. In conclusion, this work demonstrated that intratumoral injection of 5-Fu-loaded MCL may induce significant suppression of tumor growth through effective accumulation of 5-Fu in the tumor.

In vivo biocompatibility study of electrospun chitosan microfiber for tissue engineering.

In this work, we examined the biocompatibility of electrospun chitosan microfibers as a scaffold. The chitosan microfibers showed a three-dimensional pore structure by SEM. The chitosan microfibers supported attachment and viability of rat muscle-derived stem cells (rMDSCs). Subcutaneous implantation of the chitosan microfibers demonstrated that implantation of rMDSCs containing chitosan microfibers induced lower host tissue responses with decreased macrophage accumulation than did the chitosan microfibers alone, probably due to the immunosuppression of the transplanted rMDSCs. Our results collectively show that chitosan microfibers could serve as a biocompatible in vivo scaffold for rMDSCs in rats.

Stimuli-Responsive Injectable In situ-Forming Hydrogels for Regenerative Medicines

Research on injectable in situ-forming hydrogels has been conducted in diverse biomedical applications for a long period. These hydrogels exhibit sol-to-gel phase transition in accordance with the external stimuli such as temperature change. Also, unlike the traditional surgical procedures the hydrogels have the distinct properties of easy management and minimal invasiveness via simple aqueous state injections at target sites. Currently, numerous polymer materials have been reported as potential stimulus-induced in situ–forming hydrogels. In this review, a comprehensive overview of the state-of-the-art of these rapidly developing materials has been outlined. In situ–forming hydrogels formed by electrostatic and hydrophobic interactions as well as their mechanistic characteristics and biomedical applications in regenerative medicine have also been discussed. The review concludes with perspectives on the future of stimulus-induced in situ–forming hydrogels.

An Electrostatically Crosslinked Chitosan Hydrogel as a Drug Carrier

Considerable efforts have been devoted to control and maintain the sustained release of proteins. In this experiment, we used bovine serum albumin-fluorescein isothiocyanate (BSA-FITC) as a model protein to explore the potential utility of a chitosan and glycerol phosphate disodium salt (GP) hydrogel as a protein drug depot. The mixing of chitosan and GP solutions (0, 10, 20 and 30 wt%) formed a liquid at room temperature. At 37 °C, however, the chitosan/GP solutions formed hydrogels through an electrostatic crosslinking process. This electrostatic interaction between the chitosan, cationic amine group, and GP, anionic phosphate group, was confirmed by the changes of zeta potentials and particle sizes of this solution. The electrostatic interaction depended both on the GP ratios in chitosan and the incubation time of chitosan/GP solutions. Furthermore, BSA-FITC-loaded chitosan/GP hydrogels were examined for their ability as potential depots for the BSA drugs. Hence, when observed, the BSA-FITC-loaded chitosan/GP hydrogels showed an in vitro sustained release profile of BSA up to 14 days. Collectively, our results show that the chitosan/GP hydrogels described here, can serve as depots for BSA drugs.

Chitosan-based hydrogels to induce neuronal differentiation of rat muscle-derived stem cells.

In this study, we used a chitosan hydrogel as a 3-dimensional substrate for the attachment, proliferation, and differentiation of rat muscle-derived stem cells (rMDSCs) in the presence of valproic acid (VA). Chitosan solutions containing glycerol phosphate disodium salt form a hydrogel at body temperature. The chitosan hydrogel exhibited a porous 3-dimensional network that allowed the culture medium to penetrate. The chitosan hydrogel acted as a suitable biocompatible substrate for the attachment and proliferation of rMDSCs. On chitosan hydrogel in the presence of VA, rMDSCs exhibited higher expression of the neural markers, neuron-specific enolase (NSE) and beta tubulin III (Tuj-1), the oligodendrocyte marker, oligodendrocyte transcription factor 2 (Olig-2), and the astrocyte marker, glial fibrillary acidic protein (GFAP) than those in the absence of VA. Our results suggest that rMDSCs on a chitosan hydrogel in the presence of VA can differentiate into cells with a neural-like phenotype.

Injectable in situ-forming hydrogel for cartilage tissue engineering

Methoxy polyethylene glycol-poly(ε-caprolactone) (MPEG-PCL; MP) diblock copolymers undergo a solution-to-gel phase transition at body temperature and serve as ideal biomaterials for drug delivery and tissue engineering. Here, we examined the potential use of a chondrocyte-loaded MP solution as an injectable, in situ-forming hydrogel for cartilage regeneration. The chondrocyte-MP solution underwent a temperature-dependent solution-to-gel phase transition in vitro, as shown by an increase in viscosity from 1 cP at 20-30 °C to 1.6 × 105 cP at 37 °C. The chondrocytes readily attached to and proliferated on the MP hydrogel in vitro. The chondrocyte-MP solution transitioned to a hydrogel immediately after subcutaneous injection into mice, and formed an interconnected pore structure required to support the growth, proliferation, and differentiation of the chondrocytes. The chondrocyte-MP hydrogels formed cartilage in vivo, as shown by the histological and immunohistochemical staining of glycosaminoglycans, proteoglycans, and type II collagen, the major components of cartilage. Cartilage formation increased with hydrogel implantation time, and the expression of glycosaminoglycans, and type II collagen reached maximal levels at 6 weeks post-implantation. Collectively, these data suggest that in situ-forming chondrocyte-MP hydrogels have potential as non-invasive alternatives for tissue-engineered cartilage formation.