Thermo-Responsive Nanocomposite Bioink with Growth-Factor Holding and its Application to Bone Regeneration.

Three-dimensional (3D) bioprinting, which is being increasingly used in tissue engineering, requires bioinks with tunable mechanical properties, biological activities, and mechanical strength for in vivo implantation. Herein, a growth-factor-holding poly(organophosphazene)-based thermo-responsive nanocomposite (TNC) bioink system is developed. The mechanical properties of the TNC bioink are easily controlled within a moderate temperature range (5-37 °C). During printing, the mechanical properties of the TNC bioink, which determine the 3D printing resolution, can be tuned by varying the temperature (15-30 °C). After printing, TNC bioink scaffolds exhibit maximum stiffness at 37 °C. Additionally, because of its shear-thinning and self-healing properties, TNC bioinks can be extruded smoothly, demonstrating good printing outcomes. TNC bioink loaded with bone morphogenetic protein-2 (BMP-2) and transforming growth factor-beta1 (TGF-ß1), key growth factors for osteogenesis, is used to print a scaffold that can stimulate biological activity. A biological scaffold printed using TNC bioink loaded with both growth factors and implanted on a rat calvarial defect model reveals significantly improved bone regenerative effects. The TNC bioink system is a promising next-generation bioink platform because its mechanical properties can be tuned easily for high-resolution 3D bioprinting with long-term stability and its growth-factor holding capability has strong clinical applicability.

A micro-fragmented collagen gel as a cell-assembling platform for critical limb ischemia repair.

Critical limb ischemia (CLI) is a devastating disease characterized by the progressive blockage of blood vessels. Although the paracrine effect of growth factors in stem cell therapy made it a promising angiogenic therapy for CLI, poor cell survival in the harsh ischemic microenvironment limited its efficacy. Thus, an imperative need exists for a stem-cell delivery method that enhances cell survival. Here, a collagen microgel (CMG) cell-delivery scaffold (40 × 20 µm) was fabricated via micro-fragmentation from collagen-hyaluronic acid polyionic complex to improve transplantation efficiency. Culturing human adipose-derived stem cells (hASCs) with CMG enabled integrin receptors to interact with CMG to form injectable 3-dimensional constructs (CMG-hASCs) with a microporous microarchitecture and enhanced mass transfer. CMG-hASCs exhibited higher cell survival (p < 0.0001) and angiogenic potential in tube formation and aortic ring angiogenesis assays than cell aggregates. Injection of CMG-hASCs intramuscularly into CLI mice increased blood perfusion and limb salvage ratios by 40 % and 60 %, respectively, compared to cell aggregate-treated mice. Further immunofluorescent analysis revealed that transplanted CMG-hASCs have greater muscle regenerative and angiogenic potential, with enhanced cell survival than cell aggregates (p < 0.05). Collectively, we propose CMG as a cell-assembling platform and CMG-hASCs as promising therapeutics to treat CLI.

One-Step Preparation of an Injectable Hydrogel Scaffold System Capable of Sequential Dual-Growth Factor Release to Maximize Bone Regeneration.

Numerous growth factors are involved in the natural bone healing process, which is precisely controlled in a time- and concentration-dependent manner. Mimicking the secretion pattern of growth factors could be an effective means to maximize the bone regeneration effect. However, achieving the sequential delivery of various growth factors without the use of multiple materials or complex scaffold designs is challenging. Herein, an injectable poly(organophosphazene) hydrogel scaffold (IPS) encapsulating bone morphogenetic protein (BMP)-2 and TGFß-1 (IPS_BT) is studied to mimic the sequential secretion of growth factors involved in natural bone healing. The IPS_BT system is designed to release TGFß-1 slowly while retaining BMP-2 for a longer period of time. When IPS_BT is injected in vivo, the hydrogel is replaced by bone tissue. In addition, angiogenic (CD31 and alpha-smooth muscle actin (α-SMA)) and stemness (Nanog and SOX2) markers are highly upregulated in the early stages of bone regeneration. The IPS system developed here has promising applications in tissue engineering because 1) various amounts of the growth factors can be loaded in one step, 2) the release pattern of each growth factor can be controlled via differences in their molecular interactions, and 3) the injected IPS can be degraded and replaced with regenerated bone tissue.

The antitumor effect of a thermosensitive polymeric hydrogel containing paclitaxel in a peritoneal carcinomatosis model.

The prognosis of peritoneal carcinomatosis is regarded as poor because safe, effective therapeutic modalities are lacking. Intraperitoneal chemotherapy is one treatment option, involving the delivery of a high concentration of chemotherapeutic drugs into the abdominal cavity, but the severe side effects associated with such treatment are a major obstacle in clinical application. We evaluated the anti-cancer effects of intraperitoneal delivery of a thermosensitive polymeric hydrogel containing chemotherapeutics in an animal model of carcinomatosis. The progress of peritoneal carcinomatosis, introduced by injecting a luciferase-transfected human gastric cancer cell line (HSC44Luc) into the peritoneal cavity of nude mice, was quantitatively evaluated by in vivo bioluminescence imaging. Three days after intraperitoneal (IP) injection of HSC44Luc cells, treatment solutions were injected into the peritoneal cavity. Mice were categorized into four groups depending on treatment method; these were (1) a control PBS group (n = 5), (2) a hydrogel-only group (n = 5), (3) a paclitaxel solution (30 mg/kg) group (n = 3), and (4) a hydrogel-with-paclitaxel (15 mg/kg) group (n = 5). Quantitative photon counting was performed weekly in each animal. Mice were sacrificed on the 5th or 28th day after treatment, for pathologic evaluation. In vivo bioluminescence imaging showed that photon counts in the hydrogel-with-paclitaxel and paclitaxel solution groups were significantly lower than in the PBS group over the entire experimental period. Although neither group of responding mice showed any peritoneal nodules on the 28th day after treatment, only the paclitaxel solution group exhibited dilated edematous changes in the intestine; these side effects were absent in animals treated with hydrogel-with-paclitaxel group. In conclusion, a thermosensitive hydrogel containing paclitaxel may be a safe and effective treatment option for peritoneal carcinomatosis.

Long-term anti-inflammatory effects of injectable celecoxib nanoparticle hydrogels for Achilles tendon regeneration.

The treatment of chronic Achilles tendonitis (AT) often requires prolonged therapy and invasive therapeutic methods such as surgery or therapeutic endoscopy. To prevent the progression of chronic AT, excessive inflammation must be alleviated at an early stage. Corticosteroids or nonsteroidal anti-inflammatory drugs are generally prescribed to control inflammation; however, the high doses and long therapeutic periods required may lead to serious side effects. Herein, a local injectable poly(organophosphazene) (PPZ) - celecoxib (CXB) nanoparticle (PCNP) hydrogel system with long-term anti-inflammatory effects was developed for the treatment of tendonitis. The amphiphilic structure and thermosensitive mechanical properties of PPZ means that the hydrophobic CXB can be easily incorporated into the hydrophobic core to form PCNP at 4 °C. Following the injection of PCNP into the AT, PCNP hydrogel formed at body temperature and induced long-term local anti-inflammatory effects via sustained release of the PCNP. The therapeutic effects of the injectable PCNP system can alleviate excessive inflammation during the early stages of tissue damage and boost tissue regeneration. This study suggests that PCNP has significant potential as a long-term anti-inflammatory agent through sustained nonsteroidal anti-inflammatory drugs (NSAIDs) delivery and tissue regeneration boosting. STATEMENT OF SIGNIFICANCE: In the treatment of Achilles tendinitis, a long-term anti-inflammatory effect is needed to alleviate excessive inflammation and induce regeneration of the damaged Achilles tendon. Injectable poly(organophosphazene)(PPZ)-celecoxib(CXB) nanoparticles (PCNP) generated a long-term, localized-anti-inflammatory effect in the injected region, which successfully induced the expression of anti-inflammatory cytokines and suppressed pro-inflammatory cytokines, while the PCNPs degraded completely. Accordingly, regeneration of the damaged Achilles tendon was achieved through the long-term anti-inflammatory effect induced by a single PCNP injection. The PCNP system therefore has great potential in long-term NSAIDs delivery for various tissue engineering applications.

Injectable polymeric nanoparticle hydrogel system for long-term anti-inflammatory effect to treat osteoarthritis.

Treatment of osteoarthritis (OA) by administration of corticosteroids is a commonly used method in clinics using anti-inflammatory medicine. Oral administration or intra-articular injection of corticosteroids can reduce the pain and progress of cartilage degeneration, but they are usually insufficient to show local and long-term anti-inflammatory effects because of their fast clearance in the body. In this study, we suggest an injectable anti-OA drug depot system for sustained drug release that provides long-term effective therapeutic advantages. Amphiphilic poly(organophosphazene), which has temperature-dependent nanoparticle forming and sol-gel transition behaviors when dissolved in aqueous solution, was synthesized for triamcinolone acetonide (TCA) delivery. Because hydrophobic parts of the polymer can interact with hydrophobic parts of the TCA, the TCA was encapsulated into the self-assembled polymeric nanoparticles. The TCA-encapsulated polymeric nanoparticles (TePNs) were well dispersed in an aqueous solution below room temperature so that they can be easily injected as a sol state into an intra-articular region. However, the TePNs solution transforms immediately to a viscose 3D hydrogel like a synovial fluid in the intra-articular region via the conducted body temperature. An in vitro TCA release study showed sustained TCA release for six weeks. One-time injection of the TePN hydrogel system in an early stage of OA-induced rat model showed a great inhibition effect against further OA progression. The OA-induced knees completely recovered as a healthy cartilage without any abnormal symptoms.

Dual-functional hydrogel system for spinal cord regeneration with sustained release of arylsulfatase B alleviates fibrotic microenvironment and promotes axonal regeneration.

Traumatic damage to the spinal cord does not spontaneously heal, often leading to permanent tissue defects. We have shown that injection of imidazole-poly(organophosphazene) hydrogel (I-5) bridges cystic cavities with the newly assembled fibronectin-rich extracellular matrix (ECM). The hydrogel-created ECM contains chondroitin sulfate proteoglycans (CSPGs), collagenous fibrils together with perivascular fibroblasts, and various fibrotic proteins, all of which could hinder axonal growth in the matrix. In an in vitro fibrotic scar model, fibroblasts exhibited enhanced sensitivity to TGF-ß1 when grown on CSPGs. To alleviate the fibrotic microenvironment, the I-5 hydrogel was equipped with an additional function by making a complex with ARSB, a human enzyme degrading CSPGs, via hydrophobic interaction. Delivery of the I-5/ARSB complex significantly diminished the fibrotic ECM components. The complex promoted serotonergic axonal growth into the hydrogel-induced matrix and enhanced serotonergic innervation of the lumbar motor neurons. Regeneration of the propriospinal axons deep into the matrix and to the lumbar spinal cord was robustly increased accompanied by improved locomotor recovery. Therefore, our dual-functional system upgraded the functionality of the hydrogel for spinal cord regeneration by creating ECM to bridge tissue defects and concurrently facilitating axonal connections through the newly assembled ECM.

Bone Generation Following Repeated Administration of Recombinant Bone Morphogenetic Protein 2.

BACKGROUND: The delivery of recombinant human bone morphogenetic protein 2 (rhBMP2) by using various carriers has been used to successfully induce bone formation in many animal models. However, the effect of multiple administration of rhBMP2 on bone formation and BMP2 antibody production has not been determined. Our aim was to examine the bone formation activity of rhBMP2 and serum levels of anti-BMP2 antibodies following the repeated administration of rhBMP2 in mice. METHODS: Absorbable collagen sponges or polyphosphazene hydrogels containing rhBMP2 were subcutaneously implanted or injected into one side on the back of six-week-old C57BL/6 mice. Three or 4 weeks later, the same amount of rhBMP2 was administered again with the same carrier into the subcutaneous regions on the other side of the back or into calvarial defects. The effects of a single administration of rhBMP2 on the osteoinductive ability in the ectopic model were compared with those of repeated administrations. In vivo ectopic or orthotopic bone formation was evaluated using microradiography and histological analyses. Serum concentrations of anti-rhBMP2 antibodies were measured by ELISAs. RESULTS: Re-administration of the same amount of rhBMP2 into the subcutaneous area showed a comparable production of ectopic bone as after the first administration. The bone forming ability of repeated rhBMP2 administrations was equal to that of single rhBMP2 administration. The administration of rhBMP2 into calvarial defects, following the first subcutaneous administration of rhBMP2 on the back, completely recovered the defect area with newly regenerated bone within 3 weeks. Repeated administration of rhBMP2 at 4-week intervals did not significantly alter the serum levels of anti-BMP2 antibodies and did not induce any inflammatory response. The serum obtained from rhBMP2-exposed mice had no effect on the ability of rhBMP2 to induce osteogenic gene expressions in MC3T3-E1. CONCLUSION: We suggest that the osteoinductive ability of rhBMP2 is not compromised by repeated administrations. Thus, rhBMP2 can be repeatedly used for bone regeneration at various sites within a short duration.

Suppression of in vivo tumor growth by using a biodegradable thermosensitive hydrogel polymer containing chemotherapeutic agent.

Current systemic chemotherapy in the treatment of solid tumors inevitably induces various systemic adverse effects. Locally injected chemotherapy is expected to overcome this limitation of systemic therapy. We evaluated by luminescence imaging the effects of chemotherapy administered locally by means of a biodegradable thermosensitive hydrogel polymer. The human gastric cancer cell line HSC44Luc was used for tumor induction, and it was confirmed to be sensitive to doxorubicin by MTT assay. Cells were injected subcutaneously into Balb/c-nude mice. When the mean volume of tumor reached 400 mm(3), we divided the mice into 6 groups (5 per group) according to treatment: 1) control (intratumor injection of PBS), 2) systemic injection of doxorubicin, 3) intratumor injection of polymer gel, 4) intratumor injection of polymer gel physically mixed with a low dose of doxorubicin, 5) intratumor injection of polymer gel physically mixed with a high dose of doxorubicin, 6) intratumor injection of conjugated polymer gel with doxorubicin. Body weight and tumor volume were measured every 2 or 3 days for 30 days after treatment. One mouse in each group was sacrificed for histopathologic examination every week. Reductions in body weight were not significantly different among groups. The relative rate of tumor growth was 774% in Group 1, 267% in Group 2, 813% in Group 3, -186% in Group 4, and 155% in Group 6, respectively. Thus the relative rate of tumor growth in the groups treated with polymer gel mixed with doxorubicin and the groups treated with conjugated polymer gel with doxorubicin were lower than that in the control group. Locally injectable chemotherapy using a thermosensitive hydrogel polymer with doxorubicin can suppress tumor growth effectively without severe systemic toxicity.

Dual cross-linking systems of functionally photo-cross-linkable and thermoresponsive polyphosphazene hydrogels for biomedical applications.

Photo-cross-linkable, functionalized, and thermosensitive polyphosphazenes were synthesized to develop a dual cross-linking system with properties of mechanically suitable strength and controllable biodegradation for injectable biomedical applications. The aqueous solutions of the polymers exhibited sol-gel transition behaviors against temperature. The incorporated methacrylate groups were photo-cross-linked upon UV light under mild conditions, which resulted in the formation of compact three-dimensional networks. The thermoresponsive hydrophobic interactions at body temperature facilitated the rapid dual cross-linking accomplishment of the photo-cross-linking even under mild conditions. The characteristics of the polymers such as pore size and density showed that the inner three-dimensional networks depended on the degree of cross-linking of methacrylate units. Mechanical properties of the gel were also improved several folds after developing the photo-cross-linking in the network from the in vivo degradation studies. The results demonstrate that the photo-cross-linkable and thermoresponsive polyphosphazenes have great potential as injectable, biodegradable, and controllable carriers for various biomedical applications by tuning the mechanical gel property and the degradation rate.

Rapid photocrosslinkable thermoresponsive injectable polyphosphazene hydrogels.

Rapidly photocrosslinkable and thermosensitive polyphosphazene polymers have been prepared to overcome the limitations associated with long UV exposure. Short UV exposure on the thermosensitive gels under mild conditions leads to quick photocrosslinking of the acrylate groups in the polymer network, and results in a dual crosslinked network with enhanced mechanical strength. The accelerated photocrosslinking can be attributed to the high reactivity of the acrylate double bond and hydrophobic interactions in the polymer network. The effects on the degree of photocrosslinking of the UV light intensity and the concentration of the photoinitiator were studied. In vitro and in vivo photocrosslinkings were accomplished within 120 and 180 s of exposure times, respectively. The degradation rate of the polymers depended on the degree of acrylate substitution in the polymer network. These results demonstrate that the injectable hydrogels with desired mechanical properties and degradation rates can be created in situ under mild photocrosslinkable conditions, and the dual crosslinkable acrylated poly(organophosphazenes) may hold great promise for biomedical delivery applications of biological molecules, cells, and drugs.

A simple HPLC method for doxorubicin in plasma and tissues of nude mice.

Doxorubicin is a cytotoxic anthracycline that has been used for the treatment of several malignancies. Several HPLC methods have been reported for the quantification of doxorubicin in biological samples. Tissue matrix effect and sample size requirements, however, have been remaining issues for simple and easy-to-adapt analytical methods in small animal experiments. The present study established a simple HPLC method for doxorubicin in plasma and tissues (tumor, heart, spleen, liver, gastrointestinal tract, brain, lung, and kidney) of nude mice. Our method required a small sample volume (100 microL plasma and 10 mg tissue), which made it possible to use each blank tissue for calibration curves. The limit of quantification was 25 ng/mL in plasma and 0.1 to 0.4 microg/mg in other tissues with recovery rates ranging from 52.4 to 95.2%. The linearity, accuracy and precision in all tissues, except gastrointestinal tract (GIT), were found to be acceptable in the range of 25-2000 ng/mL plasma and 0.1-4 ng/mg tissue. This method was used successfully to determine the drug concentration in plasma and tissues of human tumor xenograft-bearing nude mice given intratumoral doxorubicin in a polymeric drug delivery system designed for sustained release. In conclusion, the present method may be useful as a simple and easy-to-adapt, yet, sensitive analytical method of doxorubicin for plasma and tissue pharmacokinetic studies in small animals such as nude mice.

3D hydrogel stem cell niche controlled by host-guest interaction affects stem cell fate and survival rate.

Host-guest interaction using ß-cyclodextrin (ß-CD) and adamantane (Ad) allows facile modulation of guest molecule concentration in 3D hydrogels. Based on this phenomenon, we prepared a thermosensitive poly(organophosphazene) bearing ß-CD hydrogel (ß-CD PPZ, as host) and Ad-Arg-Gly-Asp (Ad-RGD, as guest). The structures of synthesized thermosensitive ß-CD PPZ and Ad-RGD were confirmed by 1H NMR and FT-IR. The ß-CD PPZ/Ad-RGD mixture was prepared by simple mixing and elicited thermosensitive properties with the formation of gelation in all Ad-RGDs mixing proportions at the body temperature. Strong and controlled host-guest interactions between ß-CD PPZ and Ad-RGD were observed in 2D-NOESY, DLS, and TEM. Regulated MSC behaviors were elicited based on the use of controlled Ad-RGD amounts at the level of in vitro and in vivo. As the amount of Ad-RGD was increased in the ß-CD PPZ hydrogel, MSC survival rate was enhanced and was prone to express osteogenic factors. While Ad-RGD is absent or low in hydrogel, relatively poor MSC survival rate and adipogenesis were exhibited. Altogether, we verified that survival rate and differentiation of MSCs could be controlled by host-guest interaction system with thermosensitive 3D hydrogel. This proposed 3D hydrogel controlling system with host-guest interaction is expected to be a platform technology as changing guest molecules.

Fine-Tunable and Injectable 3D Hydrogel for On-Demand Stem Cell Niche.

Stem-cell-based tissue engineering requires increased stem cell retention, viability, and control of differentiation. The use of biocompatible scaffolds encapsulating stem cells typically addresses the first two problems. To achieve control of stem cell fate, fine-tuned biocompatible scaffolds with bioactive molecules are necessary. However, given that the fine-tuning of stem cell scaffolds is associated with UV irradiation and in situ scaffold gelation, this process is in conflict with injectability. Herein, a fine-tunable and injectable 3D hydrogel system is developed with the use of thermosensitive poly(organophosphazene) bearing ß-cyclodextrin (ß-CD PPZ) and two types of adamantane-peptides (Ad-peptides) that are associated with mesenchymal stem cell (MSC) differentiation and that serve as stoichiometrically controlled pendants for fine-tuning. Given that complexation of hosts and guests subject to strict stoichiometric control is achieved with simple mixing, these fabricated hydrogels exhibit well-aligned, fine-tuning responses, even in living animals. Injection of MSCs in fine-tuned hydrogels also results in various chondrogenic differentiation levels at three weeks postinjection. This is attributed to the differential controls of Ad-peptides, if MSC preconditioning is excluded. Eventually, the fine-tunable and injectable 3D hydrogel could be applied as platform technology by simply switching the types of peptides bearing adamantane and their stoichiometry.

Injectable and Quadruple-Functional Hydrogel as an Alternative to Intravenous Delivery for Enhanced Tumor Targeting.

Intravenous (IV) route is the most commonly used drug-delivery approach. However, the targeting efficiency to tumor through IV delivery is usually less than 10%. To address this limitation, we report a new systemic delivery method utilizing injectable and quadruple-functional hydrogels to improve targeting efficiency through passive, active, and magnetic targeting, and hydrogel-controlled sustained release. The hydrogels consist of a folate/polyethylenimine-conjugated poly(organophosphazene) polymer, which encapsulates small interfering RNA (siRNA) and Au-Fe3O4 nanoparticles to form a nanocapsule (NC) structure by a simple mixing. The hydrogels are localized as a long-term "drug-release depot" after a single subcutaneous injection and sol-gel phase transition. NCs released from the hydrogels enter the circulatory systems and then target the tumor through enhanced permeability and retention/folate/magnetism triple-targeting, over the course of circulation, itself prolonged by the controlled release. In vivo experiments show that 12% of NCs are successfully delivered to the tumor, which is a considerable improvement compared to most results through IV delivery. The sustained targeting of gold to tumor enables two cycles of photothermal therapy, resulting in an enhanced silencing effect of siRNA and considerable reduction of tumor volume, which we are unable to achieve via simple intravenous injection.

Sustained Release of Exendin 4 Using Injectable and Ionic-Nano-Complex Forming Polymer Hydrogel System for Long-Term Treatment of Type 2 Diabetes Mellitus.

Daily treatment of diabetes to stabilize blood glucose level poses a challenge for patients with diabetes mellitus. Diabetes is a long-term metabolic disorder, and the treatment lasts for the rest of the patient's life after diagnosis. We presented a new injectable hydrogel depot system using exendin 4 (Ex-4) interactive and complex forming polymeric ionic-nano-particles for long-term antidiabetes treatment. Protamine-conjugated polymer (ProCP) was developed to form ionic-nano-complexes with Ex-4, as the amino-group-rich protamine and the negatively charged Ex-4 ( pI: 4.86) interact with each other due to their opposite electric charges in physiological conditions. Morphologically, the ProCP were nanoparticles in aqueous condition (10 wt % of ProCP in phosphate-buffered solution, <25 °C) and formed condensed ionic- and nano-complexes with Ex-4. The complexes formed a bulk hydrogel when exposed to body temperature. A slow release of the Ex-4/ProCP ionic-nano-complexes occurred from the hydrogel depot, followed by Ex-4 dissociation from the ionic-nano-complexes and hydrolysis of ProCP. Given that the Ex-4 release occurs after the complex releases from the hydrogel, the periods of Ex-4 release and hydrogel maintenance may be similar. The present system showed a considerably prolonged Ex-4 release. Additionally, it showed potential as a long-term effective and reproducible antidiabetes treatment.

Effect of chitosan on the release of protein from thermosensitive poly(organophosphazene) hydrogels.

Poly(organophosphazenes) have been suggested as a potential thermosensitive hydrogel for use in the development of an injectable gel-depot system. Under biological conditions, hydrophilic model protein drugs, including bovine serum albumin (BSA), gelatin type B (MW 20,000) (GB20), and fluorescein isothiocyanate albumin (FITC-albumin) loaded in the hydrogels were released for 1-2 weeks, showing an initial burst release. However, this initial burst release could be suppressed when the proteins were couched in a complex with chitosan, and under these conditions evidenced a prolonged release period. BSA, GB20, and FITC-albumin, all of which are negatively charged at a pH of 7.4, interacted with chitosan harboring positive amine groups. The formation of these protein/chitosan complexes were confirmed via measurements of changes in zeta-potential and high-performance liquid chromatography. We determined the appropriate ratio of proteins to chitosan for suppression of the initial burst to be 1:5 to 1:10. From these findings, we were able to conclude that both the release rate and release period could be controlled via the formation of protein/chitosan complex.

New approach for vertical bone regeneration using in situ gelling and sustained BMP-2 releasing poly(phosphazene) hydrogel system on peri-implant site with critical defect in a canine model.

An injectable hydrogel system with sustained bone morphogenetic protein 2 (BMP-2) release ability was developed for vertical bone regeneration at peri-implant sites and enhanced osseointegration of dental implants. In three young male beagle dogs, a pair of defects was created on both sides of the mandibular bone. Next, two implants were transplanted into each defect. In situ gelling polymer solutions with or without BMP-2 were applied to cover the implants and mandibular defects. The effects of the in situ gelling and sustained BMP-2 releasing (IGSR) hydrogel system on peri-implant bone regeneration were evaluated by radiologic examination, micro-computed tomography, and histomorphometric analysis. Twelve weeks after the treatment, significant bone generation at the peri-implant site occurred following BMP-2/IGSR hydrogel treatment. Bone volume and mineral density were increased by 1.7- and 1.3-fold, respectively (p < 0.01 and 0.05 vs. control, respectively) for the BMP-2/IGSR hydrogel system. And, 0.57-0.31 mm vertical bone generation was observed at the peri-implant site for the BMP-2/IGSR hydrogel system, while rare vertical bone generation occurred in the control group. The BMP-2/IGSR hydrogel system significantly increased bone to implant contact % between induced bone and existing bone (p < 0.05 and 0.01 vs. control). These vertical bone regeneration and higher osseointegration levels demonstrated the effectiveness of the BMP-2/IGSR hydrogel system. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 751-759, 2018.

Multiple hyperthermia-mediated release of TRAIL/SPION nanocomplex from thermosensitive polymeric hydrogels for combination cancer therapy.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) possesses strong anti-cancer potential because of its ability to specifically kill cancer cells. However, clinical use of TRAIL is impeded by its short in vivo half-life and native TRIAL-resistant cancer cell populations. To overcome these limitations, we designed a multiple magnetic hyperthermia (MHT)-mediated TRAIL release system for combination therapy using an injectable, biodegradable and thermosensitive polymeric hydrogel. In this system, positively charged TRAIL and hydrophobic superparamagnetic iron oxide nanoparticles (SPIONs) are complexed with negatively charged poly(organophosphazene) polymers via ionic and hydrophobic interactions, respectively. Transmission electron microscopy images showed a nano-sized core-shell structure of the TRAIL/SPION polymeric nanocomplex in aqueous solution that transformed into a hydrogel at body temperature. Hyperthermia can enhance the release of TRAIL from hydrogels through temperature-sensitive hydrogel dissolution. TRAIL-resistant U-87 MG cells were killed by the combination of TRAIL and multiple hyperthermia via caspase-3 and -8 active apoptosis. The hyperthermia-enhanced cytotoxicity of TRAIL was dependent on the hyperthermia cycle number and corresponding TRAIL release. Significant in vivo tumor reduction was observed by combining 2 cycles of mild MHT and TRAIL release using a single injection of TRAIL/SPION nanocomplex hydrogels without damage to main organs. Furthermore, the therapeutic outcomes can be monitored by long-term magnetic resonance imaging.

Tuning physical properties and BMP-2 release rates of injectable hydrogel systems for an optimal bone regeneration effect.

For a substance to be used as a drug delivery carrier and tissue inducible material for a target disease, its drug release rate and physical properties should be optimized to facilitate the healing process. We developed multi-tunable hydrogel systems with various physical properties and release behaviors to determine the optimal conditions for bone regeneration. Five injectable poly(phosphazene) hydrogels were developed with different types and amounts of anionic side-chains. The five polymer hydrogels showed considerably different in vitro and in vivo performances for sol-gel phase transition, dissolution/degradation, water uptake, and pore size. Furthermore, bone morphogenetic protein-2 (BMP-2) was loaded into the polymer hydrogels by forming nano-sized ionic-complexes with each polymer. The five types of nanocomplex hydrogels showed completely different BMP-2 release rates. By administering each nanocomplex hydrogel to mouse calvarial, we identified the most adapted nanocomplex hydrogel system for effective bone regeneration. The BMP-2 release rate was the most important factor in effective bone regeneration. Finally, the bone regeneration effect of the optimized hydrogel system was investigated in a critical-sized calvarial defect model.