Long-term release of bone morphogenetic protein-2 (BMP-2) can promote bone regeneration. We developed an injectable system for long-term delivery of BMP-2 by covalently conjugating heparin to fibrinogen. The heparin-conjugated fibrinogen formed an injectable, heparin-conjugated fibrin (HCF) gel when mixed with thrombin. HCF released 89.4 +/- 3.8% of the loaded BMP-2 for 13 days, whereas normal fibrin released 83.7 +/- 7.6% for the initial 3 days. BMP-2 released from HCF significantly increased alkaline phosphatase activity of cultured osteoblasts, whereas BMP-2 released from normal fibrin did not do so, indicating that BMP-2 released from HCF is bioactive and suggesting that long-term delivery of BMP-2 is advantageous over short-term delivery for bone regeneration. HCF, BMP-2-loaded HCF, and BMP-2-loaded normal fibrin containing free heparin were contained in polyester cylindrical tubes and implanted into the hind limb muscle pockets of rats for 8 weeks. Soft X-ray radiography, computed tomography, histomorphometry, calcium assay, and western blot analysis showed that BMP-2-loaded HCF yielded the most extensive bone formation among the groups. Since HCF can deliver BMP-2 over a long term, is an injectable system, and is made of clinically benign materials, this system would have advantages for clinical applications to regenerate bone.

A challenge in using plasmonic nanostructure-drug conjugates for thermo-chemo combination cancer therapy lies in the huge size discrepancy; the size difference can critically differentiate their biodistributions and hamper the synergistic effect. Properly tuning the plasmonic wavelength for photothermal therapy typically results in the nanostructure size reaching ∼100 nm. We report a new combination cancer therapy platform that consists of relatively small 10 nm pH-responsive spherical gold nanoparticles and conjugated doxorubicins. They are designed to form aggregates in mild acidic environment such as in a tumor. The aggregates serve as a photothermal agent that can selectively exploit external light by their collective plasmon modes. Simultaneously, the conjugated doxorubicins are released. The spatiotemporal concertion is confirmed at the subcellular, cellular, and organ levels. Both agents colocalize in the cell nuclei. The conjugates accumulate in cancer cells by the rapid phagocytic actions and effective blockage of exocytosis by the increased aggregate size. They also effectively accumulate in tumors up to 17 times over the control because of the enhanced permeation and retention. The conjugates exhibit a synergistic effect enhanced by nearly an order of magnitude in cellular level. The synergistic effect is demonstrated by the remarkable reductions in both the therapeutically effective drug dosage and the photothermal laser threshold. Using an animal model, effective tumor growth suppression is demonstrated. The conjugates induce apoptosis to tumors without any noticeable damage to other organs. The synergistic effect in vivo is confirmed by qRT-PCR analysis over the thermal stress and drug-induced growth arrest.

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Bone morphogenetic protein-2 (BMP-2) is used to promote bone regeneration. However, the bone regeneration ability of BMP-2 relies heavily on the delivery vehicle. Previously, we have developed heparin-conjugated fibrin (HCF), a vehicle for long-term delivery of BMP-2 and demonstrated that long-term delivery of BMP-2 enhanced its osteogenic efficacy as compared to short-term delivery at an equivalent dose. The aim of this study was to compare the bone-forming ability of the BMP-2 delivered by HCF to that delivered by clinically utilized BMP-2 delivery vehicle collagen sponge. An in vitro release profile of BMP-2 showed that HCF released 80% of the loaded BMP-2 within 20 days, whereas collagen sponge released the same amount within the first 6 days. Moreover, the BMP-2 released from the HCF showed significantly higher alkaline phosphatase activity than the BMP-2 released from collagen sponge at 2 weeks in vitro. Various doses of BMP-2 were delivered with HCF or collagen sponge to mouse calvarial defects. Eight weeks after the treatment, bone regeneration was evaluated by computed tomography, histology, and histomorphometric analysis. The dose of BMP-2 delivered by HCF to achieve 100% bone formation in the defects was less than half of the BMP-2 dose delivered by collagen sponge to achieve a similar level of bone formation. Additionally, bone regenerated by the HCF-BMP-2 had higher bone density than bone regenerated by the collagen sponge-BMP-2. These data demonstrate that HCF as a BMP-2 delivery vehicle exerts better osteogenic ability of BMP-2 than collagen sponge, a clinically utilized delivery vehicle.

Implantation of ex vivo expanded and osteogenically differentiated mesenchymal stem cells (MSCs) for bone regeneration has drawbacks for clinical applications, such as poor survival of implanted cells and increased treatment expenses. As a new approach for bone regeneration that can circumvent these limitations, we propose dual delivery of substance P (SP) and bone morphogenetic protein-2 (BMP-2) to facilitate endogenous stem cell recruitment to bone defects by SP and subsequent in situ osteogenic differentiation of those cells by BMP-2. A heparin-conjugated fibrin (HCF) gel enabled dual delivery with fast release of SP and slow release of BMP-2, which would be ideal for prompt recruitment of endogenous stem cells in the first stage and time-consuming osteogenic differentiation of the recruited stem cells in the second stage. The HCF gels with SP and/or BMP-2 were implanted into mouse calvarial defects for 8 weeks. Local delivery of SP to the calvarial defects using HCF gel was more effective in recruiting MSCs to the calvarial defects than intraperitoneal or intravenous administration of SP. Many of the cells recruited by SP underwent osteogenic differentiation through local delivery of BMP-2. The efficacy of in vivo bone regeneration was significantly higher in the SP/BMP-2 dual delivery group. The dual delivery of SP and BMP-2 using the HCF gel therefore has potential as an effective bone regeneration strategy.

Bone morphogenetic proteins (BMPs) are the most potent osteoinductive growth factors. BMP-2 is clinically used for spine fusion and bone fracture healing. Commercially available BMP-2 uses a type I collagen scaffold as a carrier, but it only releases BMP-2 for a short period of time, which may release the bone formation efficacy. In the present study, we hypothesize that apatite coating of a collagen scaffold increases the release period as well as the osteogenic efficacy of BMP-2. Apatite coating was achieved by incubating collagen scaffolds in simulated body fluids (SBFs). Apatite coating on collagen scaffolds was confirmed by X-ray diffraction, electron spectroscopy for chemical analysis, attenuated total reflectance-Fourier transform infrared spectroscopy, and scanning electron microscopy. The rate and period of BMP-2 release from apatite-coated collagen scaffolds varied depending on the concentration of SBFs used. The 5× and 10× SBF apatite-coated collagen scaffolds released 91.8%±11.5% and 82.2%±13.1% of their loaded BMP-2 over 13 days in vitro, respectively, whereas noncoated collagen scaffold released 98.3%±2.2% over the initial one day. BMP-2 released from apatite-coated collagen scaffold significantly increased the alkaline phosphatase activity of cultured osteoblasts, compared with BMP-2 released from noncoated collagen scaffold. Computed tomography and histomorphometry showed that BMP-2 delivery using apatite-coated collagen scaffolds resulted in 2.5-fold higher bone formation volume and 4.0-fold higher bone formation area than BMP-2 delivery using noncoated collagen scaffolds. This study shows that simple apatite coating of a collagen scaffold results in a BMP-2 carrier that renders long-term release of BMP-2 and dramatically enhances osteogenic efficacy.

Non-porous poly(lactic-co-glycolic acid) (PLGA) microspheres have been previously proposed as an injectable scaffold for in vivo cartilage tissue engineering. In this study, we tested whether using open macroporous PLGA microspheres as an injectable scaffold for in vivo cartilage tissue engineering provides a larger surface area for cell adhesion and a larger void space for cartilage tissue regeneration and, thus, regeneration of higher quality cartilage than non-porous PLGA microspheres. Rabbit chondrocytes were mixed with non-porous or macroporous PLGA microspheres and injected immediately through 18-gauge needles into subcutaneous sites in athymic mice. Six weeks after implantation, chondrocytes implanted using both types of PLGA microspheres formed solid, white cartilaginous tissues. Histological analysis of the implants with hematoxylin and eosin, safranin O and Masson's trichrome staining confirmed cartilaginous tissue formation. The portion of cartilage tissue area in the implant cross-sectional area was significantly higher in the macroporous PLGA microsphere group than in the non-porous PLGA microsphere group (88.9% versus 34.6%, P < 0.001). Importantly, the collagen (P < 0.01) and glycosaminoglycan (P < 0.01) contents of the implants were significantly higher in the macroporous PLGA microsphere group than in the non-porous PLGA microsphere group. We conclude that an open macroporous PLGA microsphere scaffold may be useful in cartilage regeneration through minimally-invasive surgical procedures in orthopedic applications.

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