N-methyl pyrrolidone as a potent bone morphogenetic protein enhancer for bone tissue regeneration.

In medicine, N-methyl pyrrolidone (NMP) has a long track record as a constituent in medical devices approved by the Food and Drug Administration and thus can be considered as a safe and biologically inactive small chemical. In the present study, we report on the newly discovered pharmaceutical property of NMP in enhancing bone regeneration in a rabbit calvarial defect model in vivo. At the cellular level, the pharmaceutical effect of NMP was confirmed, in particular, in combination with bone morphogenetic protein (BMP)-2, because NMP increased early and late markers for maturation of preosteoblasts and human bone marrow-derived stem cells in vitro. When we used the multipotent cell line C2C12 without autologous BMP expression, NMP alone had no effect on alkaline phosphatase activity, a marker for osteogenic transdifferentiation. Nevertheless, in combination with low BMP-2 doses, alkaline phosphatase activity was more than eight times as great. Thus, the pharmaceutical NMP mode of action is that of an enhancer of BMP activity. The dependency of the effects of NMP on BMP was confirmed in preosteoblasts because noggin, an extracellular BMP inhibitor, suppressed NMP-induced increases in early markers for osteoblast maturation in vitro. At the molecular level, NMP was shown to have no effect on the binding of BMP-2 to the ectodomain of the high-affinity BMP receptor IA. However, NMP further increased the phosphorylation of p38 and Smad1,5,8 induced by BMP-2. Thus, the small chemical NMP enhances BMP activity by increasing the kinase activity of the BMP receptor complex for Smad1,5,8 and p38 and could be employed as a potent drug for bone tissue regeneration and engineering.

Functionalization of polysulfide nanoparticles and their performance as circulating carriers.

We here present an evaluation of the carrier performance of nanoparticles that are biofunctional, i.e. derivatized to provide a controlled biological activity, and environmentally responsive, since they respond to the presence of oxidants. In particular, we focus on the possibilities (a) to make the nanoparticles detectable and (b) to control their uptake in phagocytic cells, which determines their lifetime in vivo. We first describe techniques for labeling selectively the nanoparticle surface or bulk with imaging moieties (fluorophores or gold). We then show how surface composition and size, which are both controlled through the use of PEG derivatives, influence uptake by macrophages in vitro and blood circulation in vivo: for example, in vitro uptake is negligible for small (40 nm) particles but not for larger (100 nm) ones and, correspondingly, in vivo blood circulation half-life time decreases from 6.0 to 2.9 h. However, upon decoration with RGD peptides also the small particles can be significantly internalized.

Repair of critical size defects in the rat cranium using ceramic-reinforced PLA scaffolds obtained by supercritical gas foaming.

Bioresorbable scaffolds made of poly(L-lactic acid) (PLA) obtained by supercritical gas foaming were recently described as suitable for tissue engineering, portraying biocompatibility with primary osteoblasts in vitro and interesting mechanical properties when reinforced with ceramics. The behavior of such constructs remained to be evaluated in vivo and therefore the present study was undertaken to compare different PLA/ceramic composite scaffolds obtained by supercritical gas foaming in a critical size defect craniotomy model in Sprague-Dawley rats. The host-tissue reaction to the implants was evaluated semiquantitatively and similar tendencies were noted for all graft substitutes: initially highly reactive but decreasing with time implanted. Complete bone-bridging was observed 18 weeks after implantation with PLA/ 5 wt % beta-TCP (PLA/TCP) and PLA/5 wt % HA (PLA/HA) scaffolds as assessed by histology and radiography. We show here for the first time that this solvent-free technique provides a promising approach in tissue engineering demonstrating both the biocompatibility and osteoconductivity of the processed structures in vivo.

Recombinant protein-co-PEG networks as cell-adhesive and proteolytically degradable hydrogel matrixes. Part II: biofunctional characteristics.

We present here the biological performance in supporting tissue regeneration of hybrid hydrogels consisting of genetically engineered protein polymers that carry specific features of the natural extracellular matrix, cross-linked with reactive poly(ethylene glycol) (PEG). Specifically, the protein polymers contain the cell adhesion motif RGD, which mediates integrin receptor binding, and degradation sites for plasmin and matrix-metalloproteinases, both being proteases implicated in natural matrix remodeling. Biochemical assays as well as in vitro cell culture experiments confirmed the ability of these protein-PEG hydrogels to promote specific cellular adhesion and to exhibit degradability by the target enzymes. Cell culture experiments demonstrated that proteolytic sensitivity and suitable mechanical properties were critical for three-dimensional cell migration inside these synthetic matrixes. In vivo, protein-PEG matrixes were tested as a carrier of bone morphogenetic protein (rhBMP-2) to heal critical-sized defects in a rat calvarial defect model. The results underscore the importance of fine-tuning material properties of provisional therapeutic matrixes to induce cellular responses conducive to tissue repair. In particular, a lack of rhBMP or insufficient degradability of the protein-PEG matrix prevented healing of bone defects or remodeling and replacement of the artificial matrix. This work confirms the feasibility of attaining desired biological responses in vivo by engineering material properties through the design of single components at the molecular level. The combination of polymer science and recombinant DNA technology emerges as a powerful tool for the development of novel biomaterials.

In vivo targeting of dendritic cells in lymph nodes with poly(propylene sulfide) nanoparticles.

Delivery of biodegradable nanoparticles to antigen-presenting cells (APCs), specifically dendritic cells (DCs), has potential for immunotherapy. This study investigates the delivery of 20, 45, and 100nm diameter poly(ethylene glycol)-stabilized poly(propylene sulfide) (PPS) nanoparticles to DCs in the lymph nodes. These nanoparticles consist of a cross-linked rubbery core of PPS surrounded by a hydrophilic corona of poly(ethylene glycol). The PPS domain is capable of carrying hydrophobic drugs and degrades within oxidative environments. 20 nm particles were most readily taken up into lymphatics following interstitial injection, while both 20 and 45nm nanoparticles showed significant retention in lymph nodes, displaying a consistent and strong presence at 24, 72, 96 and 120h post-injection. Nanoparticles were internalized by up to 40-50% of lymph node DCs (and APCs) without the use of a targeting ligand, and the site of internalization was in the lymph nodes rather than at the injection site. Finally, an increase in nanoparticle-containing DCs (and other APCs) was seen at 96h vs. 24h, suggesting an infiltration of these cells to lymph nodes. Thus, PPS nanoparticles of 20-45nm have the potential for immunotherapeutic applications that specifically target DCs in lymph nodes.

Platelet-rich plasma and fibrin as delivery systems for recombinant human bone morphogenetic protein-2.

The aim of the present study was (1) to test whether or not platelet-rich plasma (PRP) or commercially available fibrin can increase bone regeneration compared with non-treated defects and (2) to test whether or not PRP or fibrin increases bone regeneration when used as a delivery system for recombinant human bone morphogenetic protein-2 (rhBMP-2). In 16 New Zealand White rabbits, four evenly distributed 6 mm diameter defects were drilled into the calvarial bone. The following five treatment modalities were randomly allocated to all 64 defects: (0) untreated control, (1) fibrin alone, (2) PRP alone, (3) fibrin with 15 microg rhBMP-2 and (4) PRP with 15 microg rhBMP-2. For the fibrin gels and the PRP containing rhBMP-2, the 15 microg rhBMP-2 was incorporated by precipitation within the matrices before their gelation. After 4 weeks, the animals were sacrificed and the calvarial bones were removed for histological preparation. The area fraction of newly formed bone was determined in vertical sections from the middle of the defect by applying histomorphometrical analysis. A mean area fraction of newly formed bone was found within the former defect of 23.4% (+/-13.5%) in the control sites, of 28.4% (+/-17.4%) in the fibrin sites and of 34.5% (+/-17.4%) in the PRP sites. The statistical analysis revealed no significant difference in bone formation between the three groups (ANOVA). Addition of 15 microg rhBMP-2 in the fibrin gel (59.9+/-20.3%) and the PRP gels (63.1+/-25.3%) increased bone formation significantly. No significant difference was observed between sites, where PRP or fibrin has been used as a delivery system for rhBMP-2 (ANOVA). In conclusion, the application of fibrin gels or PRP gels to bone defects is not superior to leaving the defect untreated. Regarding the amount of bone formation, the application of 15 microg rhBMP-2 in bone defects enhances the healing significantly at 4 weeks. In this animal model, commercially available fibrin and autologous PRP gels are equally effective as delivery systems for rhBMP-2.

Bone repair with a form of BMP-2 engineered for incorporation into fibrin cell ingrowth matrices.

Most growth factors naturally involved in development and regeneration demonstrate strong binding to the extracellular matrix and are retained there until being locally mobilized by cells. In spite of this feedback between cell activity and growth factor mobilization in the extracellular matrix, this approach has not been extensively explored in therapeutic situations. We present an engineered bone morphogenetic protein-2 (BMP-2) fusion protein that mimics such function in a surgically relevant matrix, fibrin, incorporated into the matrix until it is locally liberated by cell surface-associated proteases. A tripartite fusion protein, denoted TG-pl-BMP-2, was designed and produced recombinantly. An N-terminal transglutaminase substrate (TG) domain provides covalent attachment to fibrin during coagulation under the influence of the blood transglutaminase factor XIIIa. A central plasmin substrate (pl) domain provides a cleavage site for local release of the attached growth factor from the fibrin matrix under the influence of cell-activated plasmin. A C-terminal human BMP-2 domain provides osteogenic activity. TG-pl-BMP-2 in fibrin was evaluated in vivo in critical-size craniotomy defects in rats, where it induced 76% more defect healing with bone at 3 weeks with a dose of 1 mug/defect than wildtype BMP-2 in fibrin. After a dosing study in rabbits, the engineered growth factor in fibrin was evaluated in a prospective clinical study for pancarpal fusion in dogs, where it induced statistically faster and more extensive bone bridging than equivalent treatment with cancellous bone autograft. The strong healing response shown by fibrin including a bound BMP-2 variant suggests that with the combination of bound growth factor and ingrowth matrix, it may be possible to improve upon the natural growth factor and even upon tissue autograft.

Synthetic extracellular matrices for in situ tissue engineering.

Cell interactions with the extracellular matrix play important roles in guiding tissue morphogenesis. The matrix stimulates cells to influence such things as differentiation and the cells actively remodel the matrix via local proteolytic activity. We have designed synthetic hydrogel networks that participate in this interplay: They signal cells via bound adhesion and growth factors, and they also respond to the remodeling influence of cell-associated proteases. Poly(ethylene glycol)-bis-vinylsulfone was crosslinked by a Michael-type addition reaction with a peptide containing three cysteine residues, the peptide sequence being cleavable between each cysteine residue by the cell-associated protease plasmin. Cells were able to invade gel networks that contained adhesion peptides and were crosslinked by plasmin-sensitive peptides, while materials lacking either of these two characteristics resisted cell infiltration. Incorporated bone morphogenetic protein-2 (BMP-2) induced bone healing in a rat model in materials that were both adhesive and plasmin-sensitive, while materials lacking plasmin sensitivity resisted formation of bone within the material. Furthermore, when a heparin bridge was incorporated as a BMP-2 affinity site, mimicking yet another characteristic of the extracellular matrix, statistically improved bone regeneration was observed.

Bone healing in the rat and dog with nonglycosylated BMP-2 demonstrating low solubility in fibrin matrices.

A novel form of recombinant human bone morphogenetic protein-2 (BMP-2) was explored for effective incorporation and long-term retention into fibrin ingrowth matrices. The solubility of native BMP-2 is greatly dependent on its glycosylation. To enhance retention of BMP-2 in fibrin matrices, a nonglycosylated form (nglBMP-2), which is less soluble than the native glycosylated protein, was produced recombinantly and evaluated in critical-size defects in the rat calvarium (group n=6). When 1 or 20 microg nglBMP-2 was incorporated by precipitation within the matrix, 74 +/- 4% and 98 +/- 2% healing was observed in the rat calvarium, respectively, as judged radiographically by closure of the defect at 3 weeks. More soluble forms of BMP-2, used as controls, induced less healing, demonstrating a positive correlation between low solubility, retention in vitro, and healing in vivo. Subsequently, the utility of nglBMP-2 was explored in a prospective veterinary clinical trial for inter-carpal fusion in dogs, replacing the standard-of-care, namely autologous cancellous autograft, with nglBMP-2 in fibrin. In a study of 10 sequential canine patients, fibrin with 600 microg/ml nglBMP-2 performed better than autograft in the first weeks of bone healing and comparably thereafter. Furthermore, a greater fraction of animals treated with nglBMP-2 in fibrin demonstrated bone bridging across each of the treated joints at both 12 and 17 weeks than in animals treated with autograft. These results suggest that evaluation in a human clinical setting of nonglycosylated BMP-2 in fibrin matrices might be fruitful.

Repair of bone defects using synthetic mimetics of collagenous extracellular matrices.

We have engineered synthetic poly(ethylene glycol) (PEG)-based hydrogels as cell-ingrowth matrices for in situ bone regeneration. These networks contain a combination of pendant oligopeptide ligands for cell adhesion (RGDSP) and substrates for matrix metalloproteinase (MMP) as linkers between PEG chains. Primary human fibroblasts were shown to migrate within these matrices by integrin- and MMP-dependent mechanisms. Gels used to deliver recombinant human bone morphogenetic protein-2 (rhBMP-2) to the site of critical- sized defects in rat crania were completely infiltrated by cells and were remodeled into bony tissue within five weeks. Bone regeneration was dependent on the proteolytic sensitivity of the matrices and their architecture. The cell-mediated proteolytic invasiveness of the gels and entrapment of rhBMP-2 resulted in efficient and highly localized bone regeneration.