Preparation of polymeric micelles consisting of poly(propylene glycol) and poly(caprolactone).

The ring-opening polymerization of epsilon-caprolactone (CL) was carried out with polypropylene glycol (PPG) as an initiator in the presence of the monomer activator HCl. Et2O to synthesize poly(epsilon-caprolactone)-poly(propyleneglycol)-poly(epsilon-caprolactone) (PCL-PPG-PCL) triblock copolymers with change of length PPG and PCL. The micelle formation of PCL-PPG-PCL triblock copolymers in an aqueous phase was confirmed by NMR, dynamic light scattering and fluorescence techniques. The critical micelle concentration (CMC) of the PCL-PPG-PCL triblock copolymers, determined from fluorescence measurements, was in range of 1.4 x 10(-3)-4.6 x 10(-3) mg/ml with dependence on block lengths of PPG and PCL. The partition equilibrium constant, K(v), which is an indicator of the hydrophobicity of the micelles of the PCL-PPG-PCL triblock copolymers in aqueous media, was also changed with dependence on length PPG and PCL. We confirmed that the PCL-PPG-PCL triblock copolymers formed micelles and hence may be potential hydrophobic drug carriers.

Comparison of Scaffolds Fabricated via 3D Printing and Salt Leaching: In Vivo Imaging, Biodegradation, and Inflammation.

In this work, we prepared fluorescently labeled poly(ε-caprolactone-ran-lactic acid) (PCLA-F) as a biomaterial to fabricate three-dimensional (3D) scaffolds via salt leaching and 3D printing. The salt-leached PCLA-F scaffold was fabricated using NaCl and methylene chloride, and it had an irregular, interconnected 3D structure. The printed PCLA-F scaffold was fabricated using a fused deposition modeling printer, and it had a layered, orthogonally oriented 3D structure. The printed scaffold fabrication method was clearly more efficient than the salt leaching method in terms of productivity and repeatability. In the in vivo fluorescence imaging of mice and gel permeation chromatography of scaffolds removed from rats, the salt-leached PCLA scaffolds showed slightly faster degradation than the printed PCLA scaffolds. In the inflammation reaction, the printed PCLA scaffolds induced a slightly stronger inflammation reaction due to the slower biodegradation. Collectively, we can conclude that in vivo biodegradability and inflammation of scaffolds were affected by the scaffold fabrication method.

An injectable cationic hydrogel electrostatically interacted with BMP2 to enhance in vivo osteogenic differentiation of human turbinate mesenchymal stem cells.

We have designed and characterized an injectable, electrostatically bonded, in situ-forming hydrogel system consisting of a cationic polyelectrolyte [(methoxy)polyethylene glycol-b-(poly(ε-caprolactone)-ran-poly(L-lactic acid)] (MP) copolymer derivatized with an amine group (MP-NH2) and anionic BMP2. To the best of our knowledge, there have been hardly any studies that have investigated electrostatically bonded, in situ-forming hydrogel systems consisting of MP-NH2 and BMP2, with respect to how they promote in vivo osteogenic differentiation of human turbinate mesenchymal stem cells (hTMSCs). Injectable formulations almost immediately formed an electrostatically loaded hydrogel depot containing BMP2, upon injection into mice. The hydrogel features and stability of BMP2 inside the hydrogel were significantly affected by the electrostatic attraction between BMP2 and MP-NH2. Additionally, the time BMP2 spent inside the hydrogel depot was prolonged in vivo, as evidenced by in vivo near-infrared fluorescence imaging. Biocompatibility was demonstrated by the fact that hTMSCs survived in vivo, even after 8 weeks and even though relatively few macrophages were in the hydrogel depot. The osteogenic capacity of the electrostatically loaded hydrogel implants containing BMP2 was higher than that of a hydrogel that was simply loaded with BMP2, as evidenced by Alizarin Red S, von Kossa, and hematoxylin and eosin staining as well as osteonectin, osteopontin, osteocalcin, and type 1α collagen mRNA expression. The results confirmed that our injectable, in situ-forming hydrogel system, electrostatically loaded with BMP2, can enhance in vivo osteogenic differentiation of hTMSCs.

Bone regeneration by means of a three-dimensional printed scaffold in a rat cranial defect.

Recently, computer-designed three-dimensional (3D) printing techniques have emerged as an active research area with almost unlimited possibilities. In this study, we used a computer-designed 3D scaffold to drive new bone formation in a bone defect. Poly-L-lactide (PLLA) and bioactive ß-tricalcium phosphate (TCP) were simply mixed to prepare ink. PLLA + TCP showed good printability from the micronozzle and solidification within few seconds, indicating that it was indeed printable ink for layer-by-layer printing. In the images, TCP on the surface of (and/or inside) PLLA in the printed PLLA + TCP scaffold looked dispersed. MG-63 cells (human osteoblastoma) adhered to and proliferated well on the printed PLLA + TCP scaffold. To assess new bone formation in vivo, the printed PLLA + TCP scaffold was implanted into a full-thickness cranial bone defect in rats. The new bone formation was monitored by microcomputed tomography and histological analysis of the in vivo PLLA + TCP scaffold with or without MG-63 cells. The bone defect was gradually spontaneously replaced with new bone tissues when we used both bioactive TCP and MG-63 cells in the PLLA scaffold. Bone formation driven by the PLLA + TCP30 scaffold with MG-63 cells was significantly greater than that in other experimental groups. Furthermore, the PLLA + TCP scaffold gradually degraded and matched well the extent of the gradual new bone formation on microcomputed tomography. In conclusion, the printed PLLA + TCP scaffold effectively supports new bone formation in a cranial bone defect.

Effect of Drug Carrier Melting Points on Drug Release of Dexamethasone-Loaded Microspheres.

Here, we examined the effect of melting point of drug carriers on drug release of dexamethasone (Dex)-loaded microspheres. We prepared poly(L-lactide-ran-ε-caprolactone) (PLC) copolymers with varying compositions of poly(ε-caprolactone) (PCL) and poly(L-lactide) (PLLA). As the PLLA content increased, the melting points of PLC copolymers decreased from 61 to 43 °C. PLC copolymers in vials solubilized at 40-50 °C according to the incorporation of PLLA into the PCL segment. Dexamethasone (Dex)-loaded PLC (MCxLy) microspheres were prepared by the oil-in-water (O/W) solvent evaporation/extraction method. The preparation yields were above 70%, and the mean particle size ranged from 30 to 90 µm. The MCxLy microspheres also showed controllable melting points in the range of 40-60 °C. Dex-loaded MCxLy microspheres showed similar in vitro and in vivo sustained release patterns after the initial burst of Dex. The in vitro and in vivo order of the Dex release was MC80L20 > MC90L10 > MC95L5, which agreed well with the melting point order of the drug carrier. Using in vivo fluorescence imaging of fluorescein (FI)-loaded microspheres implanted in animals, we confirmed the sustained release of FI over an extended period. In vivo inflammation associated with the PLC microsphere implants was less pronounced than that associated with Poly(lactide-co-glycolide) (PLGA). In conclusion, we successfully demonstrated that it is possible to control Dex release using Dex-loaded MCxLy microspheres with different melting points.

Synergistic anti-tumor activity through combinational intratumoral injection of an in-situ injectable drug depot.

Here, we describe combinational chemotherapy via intratumoral injection of doxorubicin (Dox) and 5-fluorouracil (Fu) to enhance the efficacy and reduce the toxicity of systemically administered Fu and Dox in cancer patients. As the key concept in this work, mixture formulations of Dox-loaded microcapsules (Dox-M) and Fu-loaded Pluronic(®) hydrogels (Fu-HP) or Fu-loaded diblock copolymer hydrogels (Fu-HC) have been employed as drug depots. The in vitro and in vivo drug depot was designed as a formulation of Dox-M dispersed inside an outer shell of Fu-HP or Fu-HC after injection. The Dox-M/Fu-HP and Dox-M/Fu-HC formulations are free flowing at room temperature, indicating injectability, and formed a structural gelatinous depot in vitro and in vivo at body temperature. The Fu-HP, Fu-HC, Dox-M/Fu-HP, Dox-M/Fu-HC, and Dox-M formulations were easily injected into tumor centers in mice using a needle. Dox-M/Fu-HC produced more significant inhibitory effects against tumor growth than that by Dox-M/Fu-HP, while Fu-HP, Fu-HC and Dox-M had the weakest inhibitory effects of the tested treatments. The in vivo study of Dox and Fu biodistribution showed that high Dox and Fu concentrations were maintained in the target tumor only, while distribution to normal tissues was not observed, indicating that Dox and Fu concentrations below their toxic plasma concentrations should not cause significant systemic toxicity. The Dox-M/Fu-HP and Dox-M/Fu-HC drug depots described in this work showed excellent performance as chemotherapeutic delivery systems. The results reported here indicate that intratumoral injection using combination chemotherapy with Dox-M/Fu-HP or Dox-M/Fu-HC could be of translational research by enhancing the synergistic inhibitory effects of Dox and Fu on tumor growth, while reducing their systemic toxicity in cancer patients.

Biodegradable poly(lactide-co-glycolide-co-ε-caprolactone) block copolymers - evaluation as drug carriers for a localized and sustained delivery system.

To develop an appropriate drug carrier for drug delivery systems, we prepared random poly(lactide-co-glycolide-co-ε-caprolactone) (PLGC) copolymers in comparison to commercial poly(lactic acid-co-glycolic acid) (PLGA) grades. The molecular weights of PLGC copolymers varied from 20k to 90k g mol-1 in the total polyester segments, when poly-l-lactic acid (PLLA), polyglycolic acid (PGA), and polycaprolactone (PCL) compositions were kept constant. The lengths of PLGC copolymers varied from 10 : 10 : 80 to 40 : 40 : 20 in the PLLA : PGA : PCL segments, when the molecular weights of the total polyester segments were kept constant. The crystalline properties of the PLGA copolymers can be changed to amorphous by the incorporation of PCL segments. In vitro and in vivo degradation behavior can be easily tuned from a few days to a few weeks by changing the chemical composition of the PLGC copolymers. The in vivo inflammation associated with the PLGC implants was less pronounced than that associated with PLGA. In this study, as drug delivery carriers for locally implantable paclitaxel (Ptx) dosages, Ptx-loaded PLGC and PLGA films showed in vitro and in vivo Ptx release for 35 days. The orders of Ptx release showed profiles similar to those of in vitro and in vivo degradation of PLGC. Using near-infrared (NIR) fluorescence imaging, we confirmed the sustained release of NIR over an extended period from IR-780-loaded PLGC and PLGA implanted in live animals. In conclusion, we confirmed that compared to PLGA, PLGC effectively acts as a drug carrier for drug delivery systems.

Evaluation of small intestine submucosa and poly(caprolactone-co-lactide) conduits for peripheral nerve regeneration.

The present study employed nerve guidance conduits (NGCs) only, which were made of small intestine submucosa (SIS) and poly(caprolactone-co-lactide) (PCLA) to promote nerve regeneration in a peripheral nerve injury (PNI) model with nerve defects of 15 mm. The SIS- and PCLA-NGCs were easily prepared by rolling of a SIS sheet and a bioplotter using PCLA, respectively. The prepared SIS- and PCLA-NGCs fulfilled the general requirement for use as artificial peripheral NGCs such as easy fabrication, reproducibility for mass production, suturability, sterilizability, wettability, and proper mechanical properties to resist collapsing when applied to in vivo implantation. The SIS- and PCLA-NGCs appeared to be well integrated into the host sciatic nerve without causing dislocations and serious inflammation. All NGCs stably maintained their NGC shape for 8 weeks without collapsing, which matched well with the nerve regeneration rate. Staining of the NGCs in the longitudinal direction showed that the regenerated nerves grew successfully from the SIS- and PCLA-NGCs through the sciatic nerve-injured gap and connected from the proximal to distal direction along the NGC axis. SIS-NGCs exhibited a higher nerve regeneration rate than PCLA-NGCs. Collectively, our results indicate that SIS- and PCLA-NGCs induced nerve regeneration in a PNI model, a finding that has significant implications in the future with regard to the feasibility of clinical nerve regeneration with SIS- and PCLA-NGCs prepared through an easy fabrication method using promising biomaterials.

A computer-designed scaffold for bone regeneration within cranial defect using human dental pulp stem cells.

A computer-designed, solvent-free scaffold offer several potential advantages such as ease of customized manufacture and in vivo safety. In this work, we firstly used a computer-designed, solvent-free scaffold and human dental pulp stem cells (hDPSCs) to regenerate neo-bone within cranial bone defects. The hDPSCs expressed mesenchymal stem cell markers and served as an abundant source of stem cells with a high proliferation rate. In addition, hDPSCs showed a phenotype of differentiated osteoblasts in the presence of osteogenic factors (OF). We used solid freeform fabrication (SFF) with biodegradable polyesters (MPEG-(PLLA-co-PGA-co-PCL) (PLGC)) to fabricate a computer-designed scaffold. The SFF technology gave quick and reproducible results. To assess bone tissue engineering in vivo, the computer-designed, circular PLGC scaffold was implanted into a full-thickness cranial bone defect and monitored by micro-computed tomography (CT) and histology of the in vivo tissue-engineered bone. Neo-bone formation of more than 50% in both micro-CT and histology tests was observed at only PLGC scaffold with hDPSCs/OF. Furthermore, the PLGC scaffold gradually degraded, as evidenced by the fluorescent-labeled PLGC scaffold, which provides information to tract biodegradation of implanted PLGC scaffold. In conclusion, we confirmed neo-bone formation within a cranial bone defect using hDPSCs and a computer-designed PLGC scaffold.

In vivo osteogenic differentiation of human turbinate mesenchymal stem cells in an injectable in situ-forming hydrogel.

Human turbinate mesenchymal stromal cells (hTMSCs) are an alternate source of adult stem cells for regenerative medicine. In this work, we demonstrated that hTMSCs are easily harvested from turbinate tissue using a minimal surgical procedure. hTMSCs showed positive expression of mesenchymal stem cell markers and proliferated at a high rate. The specific surface proteins of harvested hTMSCs were relatively tolerant of ex vivo manipulation in culture. hTMSCs exhibited osteogenic differentiation in vitro in the presence of osteogenic factors. To examine osteogenic differentiation of hTMSCs in vivo in an injectable hydrogel, cells were incorporated into a methoxy polyethylene glycol-polycaprolactone block copolymer (MPEG-PCL (MP)) solution simply by mixing. hTMSC-loaded MP solutions exhibited a temperature-dependent solution-to-gel phase transition. The hTMSC attached and grew well on in vitro- and in vivo-formed MP hydrogels. hTMSC-loaded MP solutions formed a hydrogel almost immediately upon injection into animals and the cells remained viable, even after 12 weeks. Injected hTMSCs in in situ-formed MP hydrogels differentiated into osteogenic cells, mainly in the presence of osteogenic factors. Differentiated osteoblasts were identified by Alizarin Red S, von Kossa, and alkaline phosphatase (ALP) staining, and osteonectin, osteopontin, and osteocalcin mRNA expression. To the best of our knowledge, this is the first study to show hTMSCs undergoing osteogenic differentiation in in vivo-formed MP hydrogels. In conclusion, hTMSCs could serve as adult stem cell sources and, when embedded in an in situ-formed hydrogel, may provide numerous benefits as a noninvasive alternative for bone tissue engineering applications.

Preparation of three-dimensional scaffolds by using solid freeform fabrication and feasibility study of the scaffolds.

To adapt biomaterials for solid freeform fabrication (SFF), methoxy polyethylene glycol (MPEG)-(PLLA-co-PCL) (LxCy) block copolymers were prepared using MPEG as the initiator to precisely control the molecular weight of PLLA and PCL. The LxCy block copolymers were designed such that the PLLA and PCL content varied and their molecular weights were within 200-1000 kDa. The cylindrical LxCy scaffolds were prepared by using LxCy block copolymers in SFF. The feasibility of using LxCy block copolymers was examined in terms of flowability from the micronozzle and solidification at room temperature after scaffold printing. The flowability and solidification of LxCy block copolymers mainly depend on the proportions of PLLA and PCL. Fabrication of the cylindrical LxCy scaffolds by using SFF was rapid and showed high reproducibility. In in vivo implantation, the cylindrical LxCy scaffolds exhibited biocompatibility and gradual biodegradation on a 16 week timescale. Immunohistochemical characterization showed that the in vivo LxCy scaffolds elicit only a modest inflammatory response. Taken together, these results show that LxCy block copolymers may serve as suitable biomaterials for the fabrication of well-defined three-dimensional scaffolds by using SFF.

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.

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.

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.