Skull bone regeneration in nonhuman primates by controlled release of bone morphogenetic protein-2 from a biodegradable hydrogel

The objective of this study was to investigate the feasibility of biodegradable gelatin hydrogels as the controlled-release carrier of bone morphogenetic protein-2 (BMP-2) to enhance bone regeneration at a skull defect of nonhuman primates. Hydrogels with 3 different water contents were prepared through glutaraldehyde crosslinking of gelatin with an isoelectric point of 9.0 under varied reaction conditions. A critical-sized defect (6 mm in diameter) was prepared at the skull bone of skeletally mature cynomolgus monkeys, and gelatin hydrogels incorporating various doses of BMP-2 were applied to the defects. When the bone regeneration was evaluated by soft radiography and bone mineral density (BMD) examinations, the gelatin hydrogel incorporating BMP-2 exhibited significantly higher osteoinduction activity than did an insoluble bone matrix that incorporated BMP-2 (one of the best osteoinduction systems), although the activity depended on the water content of hydrogels. BMD enhancement was highest for the gelatin hydrogel that had a water content of 97.8 wt% among all types of hydrogels. Moreover, the gelatin hydrogel enabled BMP-2 to induce the bone regeneration in nonhuman primates even at low doses. We conclude that the controlled release of BMP-2 for a certain time period was essential to inducing the osteoinductive potential of BMP-2.     

Enhanced osteoinduction by controlled release of bone morphogenetic protein-2 from biodegradable sponge composed of gelatin and beta-tricalcium phosphate

Biodegradable gelatin sponges at different contents of beta-tricalcium phosphate (beta-TCP) were fabricated to allow bone morphogenetic protein (BMP)-2 to incorporate into them. The in vivo osteoinduction activity of the sponges incorporating BMP-2 was investigated, while their in vivo profile of BMP-2 release was evaluated. The sponges prepared had an interconnected pore structure with an average pore size of 200 microm, irrespective of the beta-TCP content. The in vivo release test revealed that BMP-2 was released in vivo at a similar time profile, irrespective of the beta-TCP content. The in vivo time period of BMP-2 retention was longer than 28 days. When the osteoinduction activity of gelatin or gelatin-beta-TCP sponges incorporating BMP-2 was studied following the implantation into the back subcutis of rats in terms of histological and biochemical examinations, homogeneous bone formation was histologically observed throughout the sponges, although the extent of bone formation was higher in the sponges with the lower contents of beta-TCP. On the other hand, the level of alkaline phosphatase activity and osteocalcin content at the implanted sites of sponges decreased with an increase in the content of beta-TCP. The gelatin sponge exhibited significantly higher osteoinduction activity than that of any gelatin-beta-TCP sponge, although every sponge with or without beta-TCP showed a similar in vivo profile of BMP-2 release. In addition, the in vitro collagenase digestion experiments revealed that the gelatin-beta-TCP sponge collapsed easier than the gelatin sponge without beta-TCP incorporation. These results suggest that the maintenance of the intrasponge space necessary for the osteoinduction is one factor contributing to the osteoinduction extent of BMP-2-incorporating sponges.     

Improving bone formation in a rat femur segmental defect by controlling bone morphogenetic protein-2 release

Nonunion is a common complication in open fractures and other severe bone injuries. Recombinant human bone morphogenetic protein-2 (rhBMP-2) delivered on a collagen sponge enhances healing of fractures. However, the burst release of rhBMP-2 necessitates supra-physiological doses of rhBMP-2 to achieve a robust osteogenic effect, which introduces risk of ectopic bone formation and severe inflammation and increases the cost. Although the concept that the ideal pharmacokinetics for rhBMP-2 includes both a burst and sustained release is generally accepted, investigations into the effects of the release kinetics on new bone formation are limited. In the present study, biodegradable polyurethane (PUR) and PUR/microsphere [PUR/poly(lactic-co-glycolic acid)] composite scaffolds with varying rhBMP-2 release kinetics were compared to the collagen sponge delivery system in a critical-sized rat segmental defect model. Microcomputed tomography analysis indicated that a burst followed by a sustained release of rhBMP-2 from the PUR scaffolds regenerated 50% more new bone than the collagen sponge loaded with rhBMP-2, whereas a sustained release without the burst did not form significantly more bone than the scaffold without rhBMP-2. This study demonstrated that the putative optimal release profile (i.e., burst followed by sustained release) for rhBMP-2 can be achieved using PUR scaffolds, and that this enhanced pharmacokinetics regenerated more bone than the clinically available standard of care in a critical-sized defect in rat femora.     

Segmental bone regeneration using a load-bearing biodegradable carrier of bone morphogenetic protein-2

Segmental defect regeneration has been a clinical challenge. Current tissue-engineering approach using porous biodegradable scaffolds to delivery osteogenic cells and growth factors demonstrated success in facilitating bone regeneration in these cases. However, due to the lack of mechanical property, the porous scaffolds were evaluated in non-load bearing area or were stabilized with stress-shielding devices (bone plate or external fixation). In this paper, we tested a scaffold that does not require a bone plate because it has sufficient biomechanical strength. The tube-shaped scaffolds were manufactured from poly(propylene) fumarate/tricalcium phosphate (PPF/TCP) composites. Dicalcium phosphate dehydrate (DCPD) were used as bone morphogenetic protein-2 (BMP-2) carrier. Twenty-two scaffolds were implanted in 5mm segmental defects in rat femurs stabilized with K-wire for 6 and 15 weeks with and without 10 microg of rhBMP-2. Bridging of the segmental defect was evaluated first radiographically and was confirmed by histology and micro-computer tomography (microCT) imaging. The scaffolds in the BMP group maintained the bone length throughout the duration of the study and allow for bridging. The scaffolds in the control group failed to induce bridging and collapsed at 15 weeks. Peripheral computed tomography (pQCT) showed that BMP-2 does not increase the bone mineral density in the callus. Finally, the scaffold in BMP group was found to restore the mechanical property of the rat femur after 15 weeks. Our results demonstrated that the load-bearing BMP-2 scaffold can maintain bone length and allow successfully regeneration in segmental defects.     

Enhanced regeneration of critical bone defects using a biodegradable gelatin sponge and beta-tricalcium phosphate with bone morphogenetic protein-2

We examine the osteogenicity of a sponge biomaterial consisting of a biodegradable mixture of gelatin and beta-tricalcium phosphate (betaTCP) that bound bone morphogenetic protein 2 (BMP-2) in critical-sized bone defects in rats. Gelatin-betaTCP sponges containing either phosphate buffered saline or incorporating BMP-2 are implanted into 5 mm diameter bone defects created in rat mandibles. We assess the defects biweekly for 8 weeks following implantation. There is significantly higher osteoinductive activity and significantly more Gla-osteocalcin content at bone-defect healing sites treated with gelatin-betaTCP sponges incorporating BMP-2 than there is in those treated with sponges that did not contain BMP-2. Histologically, new bone that contains bone marrow and that is connected to the original bone almost entirely replaces the regenerated bone. These results show that biodegradable gelatin-betaTCP incorporating BMP-2 is osteogenic enough to promote healing in large bone defects.     

Bone regeneration by recombinant human bone morphogenetic protein-2 and a novel biodegradable carrier in a rabbit ulnar defect model

The effects of recombinant human bone morphogenetic protein (rhBMP)-2 and a novel carrier, PLGA-coated gelatin sponge (PGS), on bone defect repair was examined. A 1.5 cm unilateral segmental bone defect was created in the ulnar diaphysis of a Japanese white rabbit. In an initial study, defects were either treated with PGS impregnated with various concentrations of rhBMP-2 (0, 0.1, 0.4 and 1 mg/cm(3)) or left untreated. Defect healing was assessed by radiographic union rate, and biomechanical properties of regenerated bones were determined at 16 weeks postoperatively. In a second study, defects were implanted with PGS with or without rhBMP-2, and histologically observed at postoperative weeks 8 and 16. Radiographic union rate increased the dose-dependently at an early time point. All defects treated with rhBMP-2 (0.4 and 1 mg/cm(3)) were radiographically repaired. Mechanical properties of regenerated bones were restored in a dose-dependent manner. Neither ulnae left untreated nor implanted PGS alone showed radiographic union. Longitudinal alignment of lamellar structure was observed histologically at 16 weeks, indicating that remodeling of regenerated bone was complete. Implanted PGS was almost completely resorbed by 8 weeks, and no abnormalities were observed in the surrounding soft tissue. These results suggest that PGS is a promising carrier for rhBMP-2.     

Stimulation of bone regeneration following the controlled release of water-insoluble oxysterol from biodegradable hydrogel

Recently bone graft substitutes using bone morphogenetic proteins (BMPs) have been heralded as potential alternatives to traditional bone reconstruction procedures. BMP-based products, however, are associated with significant and potentially life-threatening side effects when used in the head and neck region and furthermore, are exorbitantly priced. Oxysterols, products of cholesterol oxidation, represent a class of molecules that are favorable alternatives or adjuncts to BMP therapy due to their low side effect profile and cost. In order to establish the optimal clinical utility of oxysterol, an optimal scaffold must be developed, one that allows the release of oxysterol in a sustained and efficient manner. In this study, we prepare a clinically applicable bone graft substitute engineered for the optimal release of oxysterol. We first solubilized oxysterol in water by making use of polymeric micelles using l-lactic acid oligomer (LAo) grafted gelatin. Then, the water-solubilized oxysterol was incorporated into a biodegradable hydrogel that was enzymatically degraded intracorporeally. In this manner, oxysterol could be released from the hydrogel in a degradation-driven manner. The water-solubilized oxysterol incorporated biodegradable hydrogel was implanted into rat calvarial defects and induced successful bone regeneration. The innovative significance of this study lies in the development of a bone graft substitute that couples the osteogenic activity of oxysterol with a scaffold designed for optimized oxysterol release kinetics, all of which lead to better repair of bone defects.     

Restoration of segmental bone defects in rabbit radius by biodegradable capsules containing recombinant human bone morphogenetic protein-2

Recombinant human bone morphogenetic protein-2 (rhBMP-2) was encapsulated in biodegradable poly(DL-lactide-co-glycolide) (PLGA) capsules to regenerate bone by controlling the release rate of rhBMP-2. The rhBMP-2/PLGA capsules containing 12 microg of rhBMP-2 were implanted in seven 15-mm segmental defects of rabbits radii to examine the healing capacity of the rhBMP-2/PLGA capsules. For the control group, four segmental defects were left empty and two were implanted with ghost PLGA capsules. Healing of the defects was followed for 24 weeks and periodically evaluated by radiographs and histological examination. Mechanical testing was applied to three regenerated bone samples at 24 weeks postoperatively when the mature cortex was observed. Mechanical properties of regenerated bone were not significantly different from normal intact bone statistically. Histologically, the rhBMP-2/PLGA capsules disappeared completely during the process of bone regeneration. These results increased possibilities for clinical application of rhBMP-2/PLGA capsules.     

Hollow hydroxyapatite microspheres: A novel bioactive and osteoconductive carrier for controlled release of bone morphogenetic protein-2 in bone regeneration

The regeneration of large bone defects is a common and significant clinical problem. Limitations associated with existing treatments such as autologous bone grafts and allografts have increased the need for synthetic bone graft substitutes. The objective of this study was to evaluate the capacity of novel hollow hydroxyapatite (HA) microspheres to serve as a carrier for controlled release of bone morphogenetic-2 (BMP2) in bone regeneration. Hollow HA microspheres (106–150 μm) with a high surface area (>100 m² g^?1) and a mesoporous shell wall (pore size 10–20 nm) were created using a glass conversion technique. The release of BMP2 from the microspheres into a medium composed of diluted fetal bovine serum in vitro was slow, but it occurred continuously for over 2 weeks. When implanted in rat calvarial defects for 3 or 6 weeks, the microspheres loaded with BMP2 (1 μg per defect) showed a significantly better capacity to regenerate bone than those without BMP2. The amount of new bone in the defects implanted with the BMP2-loaded microspheres was 40% and 43%, respectively, at 3 and 6 weeks, compared to 13% and 17%, respectively, for the microspheres without BMP2. Coating the BMP2-loaded microspheres with a biodegradable polymer, poly(lactic-co-glycolic acid), reduced the amount of BMP2 released in vitro and, above a certain coating thickness, significantly reduced bone regeneration in vivo. The results indicate that these hollow HA microspheres could provide a bioactive and osteoconductive carrier for growth factors in bone regeneration.     

Enhanced osteogenic activity of bone morphogenetic protein-2 by 2-O-desulfated heparin

The objective of this study was to investigate the effect of heparin sulfate groups on the osteogenic activity of bone morphogenetic protein-2 (BMP-2) in vitro and in vivo. Three types of desulfated (DS) derivatives of heparin (2-O-DS, 6-O-DS, and N-DS) were prepared and their bioactivity in rat bone marrow derived mesenchymal stem cells (MSC) in the absence or presence of BMP-2 was evaluated. When cultured with the 2-O-DS derivative and BMP-2 MSC showed enhanced proliferation, alkaline phosphatase activity, and Runx2 mRNA expression, compared with heparin and other derivatives. A similar tendency was observed for MSC cultured on two-dimensional substrates coated with heparin or the derivatives and in three-dimensional hydrogels containing heparin or the derivatives. A binding experiment demonstrated a greater binding affinity of 2-O-DS for BMP-2 than that of heparin and the other derivatives. Following implantation into the back subcutis of mice significantly greater ectopic bone formation in terms of bone weight, amount of calcium, and histology were observed for the gelatin hydrogels incorporating 2-O-DS and containing BMP-2. In addition, the gelatin hydrogels incorporating 2-O-DS showed controlled release of BMP-2 in vitro and in vivo. These findings demonstrated that the 2-O-DS derivative of heparin has a synergistic effect on the in vitro and in vivo osteogenic activity of BMP-2.     

Heparin-decorated, hyaluronic acid-based hydrogel particles for the controlled release of bone morphogenetic protein 2

We are interested in developing hydrophilic particulate systems that are capable of sequestering growth factors, regulating their release and potentiating their biological functions. To this end heparin (HP)-decorated, hyaluronic acid (HA)-based hydrogel particles (HGPs) were synthesized using an inverse emulsion polymerization technique employing divinyl sulfone as the crosslinker. By varying the feed composition of the aqueous phase the amount of HP integrated in the particles can be systematically tuned. The resulting microscopic particles are spherical in shape and contain nanosized pores suitable for growth factor encapsulation. The covalently immobilized HP retained its ability to bind bone morphogenetic protein-2 (BMP-2) specifically, and its release kinetics can be adjusted by tuning the particle composition. Compared with pure HA particles the hybrid HA/HP HGPs show a higher BMP-2 loading capacity. While BMP-2 was released from HA HGPs with a significant initial burst, a near zero order release kinetics was observed from HA/HP hybrid particles with an optimized heparin content of 0.55 μg per mg HGPs. The ability of HA/HP hybrid particles to present BMP-2 in a controlled manner, combined with the innate bioactivity of HA, induced robust and consistent chondrogenic differentiation of murine mesenchymal stem cells, as shown by up-regulation of the mRNA levels of chondrogenic markers and the production of cartilage-specific extracellular matrix components. The simplicity of the particle synthesis, combined with the defined biological activities of the constituent building blocks, renders the HP-decorated, HA-based hydrogel particle system an attractive candidate for the sustained release of BMP-2, possibly for cartilage repair and regeneration.     

Comparison of bone regeneration in a rabbit skull defect by recombinant human BMP-2 incorporated in biodegradable hydrogel and in solution

The objective of this study is to compare bone regeneration induced by recombinant human bone morphogenetic protein-2 (rhBMP-2) incorporated into a biodegradable gelatin hydrogel with that by rhBMP-2 in aqueous solution. After treating rabbit skull defects of 6 mm diameter with the two rhBMP-2 dosage forms, both of them increased the bone mineral density (BMD) at the skull defects with implantation time to a significantly higher extent than a rhBMP-2-free aqueous solution and a rhBMP-2-free empty gelatin hydrogel (p < 0.05). There was no quantifiable difference in BMD between the two dosage forms of rhBMP-2. Histological examination revealed that the integrity of newly generated bone increased with the rhBMP-2 dose, irrespective of the dosage form. The bone defect was filled with regenerated bone 21 days after treatment.     

Biodegradable gelatin microparticles as delivery systems for the controlled release of bone morphogenetic protein-2

This work evaluated gelatin microparticles and biodegradable composite scaffolds for the controlled release of bone morphogenetic protein-2 (BMP-2) in vitro and in vivo. Gelatin crosslinking (10 and 40mM glutaraldehyde), BMP-2 dose (6 and 60ng BMP-2 per mg dry microparticles), buffer type (phosphate buffered saline (PBS) and collagenase-containing PBS), and gelatin type (acidic and basic) were investigated for their effects on BMP-2 release. Release profiles were also observed using poly(lactic-co-glycolic acid) (PLGA) microparticles with varying molecular weights (8300 and 57,500). In vitro and in vivo studies were conducted using radiolabeled BMP-2; the chloramine-T method was preferred over Bolton-Hunter reagent for radioiodination with this system. BMP-2 release from PLGA microparticles resulted in a moderate burst release followed by minimal cumulative release, while BMP-2 release from gelatin microparticles exhibited minimal burst release followed by linear release kinetics in vitro. Growth factor dose had a small effect on its normalized release kinetics probably because of an equilibrium between gelatin-bound and unbound BMP-2. Differences in release from acidic and basic gelatin microparticles may result from the different pretreatment conditions used for gelatin synthesis. The in vitro release kinetics for both gelatin microparticles alone and within composite scaffolds were dependent largely on the extent of gelatin crosslinking; varying buffer type served to confirm that controlled release relies on enzymatic degradation of the gelatin for controlled release. Finally, in vivo studies with composite scaffolds exhibited minimal burst and linear release up to 28 days. In summary, dose effects on BMP-2 release were found to be minimal while varying gelatin type and release medium can alter release kinetics. These results demonstrate that a systematic control of BMP-2 delivery from gelatin microparticles can be achieved by altering the extent of basic gelatin crosslinking.     

Enhanced bone morphogenetic protein-2 performance on hydroxyapatite ceramic surfaces

The immobilization of biomolecules on biomaterial surfaces allows for the control of their localization and retention. In numerous studies, proteins have been simply adsorbed to enhance the biological performance of various materials in vivo. We investigated the potential of surface modification techniques on hydroxyapatite (HA) ceramic discs in an in vitro approach. A novel method for protein immobilization was evaluated using the aminobisphosphonates pamidronate and alendronate, which are strong Ca chelating agents, and was compared with the established silanization technique. Lysozyme and bone morphogenetic protein-2 (BMP-2) were used to assess the suitability of the two surface modification methods with regard to the enzymatic activity of lysozyme and to the capacity of BMP-2 to stimulate the osteoblastic differentiation of C2C12 mouse myoblasts. After immobilization, a 2.5-fold increase in enzymatic activity of lysozyme was observed compared with the control. The alkaline phosphatase activity per cell stimulated by immobilized BMP-2 was 2.5-fold higher [9 x 10(-6) I.U.] than the growth factor on unmodified surfaces [2-4 x 10(-6) I.U.]. With regard to the increase in protein activity, both procedures lead to equivalent results. Thus, the bisphosphonate-based surface modification represents a safe and easy alternative for the attachment of proteins to HA surfaces.     

Synergistic effects of the dual release of stromal cell-derived factor-1 and bone morphogenetic protein-2 from hydrogels on bone regeneration

The objective of this study is to evaluate the activity of gelatin hydrogels incorporating combined stromal cell-derived factor-1 (SDF-1) and bone morphogenetic protein-2 (BMP-2) on the in vivo bone regeneration at an ulna critical-sized defect and subcutaneous site of rats, and compared with that of those incorporating either SDF-1 or BMP-2. The similar release profile of SDF-1 and BMP-2 from the hydrogels was observed with or without the combination of BMP-2 and SDF-1, respectively. An enhanced bone regeneration by the hydrogels incorporating combined SDF-1 and BMP-2 was observed. In addition, the implantation of hydrogels incorporating combined SDF-1 and BMP-2 enhanced the expression level of CXC chemokine cell-surface receptor-4 (Cxcr4), Runt-related factor-2 (Runx2), and Osteocalcin genes. The experiments with green fluorescent protein (GFP)-positive Chimeric mice revealed that the recruitment of bone marrow-derived cells was promoted and a vascular-like structure together with strong accumulation of CD31- and CD34-positive cells was observed at the site of hydrogels incorporating combined SDF-1 and BMP-2 implanted. In addition, a large fraction of CD29- and CD44-positive non-hematopoietic cells was detected. It is concluded that the combined release of SDF-1 and BMP-2 enhanced the recruitment of osteogenic cells and angiogenesis, resulting in the synergistic effect on bone regeneration.     

Homogeneous osteogenesis and bone regeneration by demineralized bone matrix loading with collagen-targeting bone morphogenetic protein-2

Considerable research has been focused on the development of bone morphogenetic protein-2 (BMP-2) delivery system for homologous and efficient bone regeneration. The aim of the present study was to develop a collagen-based targeting bone repair system. A collagen-binding domain (CBD) was added to the N-terminal of native BMP-2 to allow it bind to collagen specifically. We showed that the collagen-binding bone morphogenetic protein-2 (named bone morphogenetic protein2-h, BMP2-h) had maintained the full biological activity as compared to rhBMP2 lacking the CBD. In vitro functional study also demonstrated that collagen matrix could maintain higher bioactivity of BMP2-h than native BMP-2. When demineralized bone matrix (DBM) impregnated with BMP2-h was implanted subcutaneously in rats, homogeneous bone formation was observed. Moreover, in a rabbit mandible defect model, surgical implantation of collagen matrix loaded with BMP2-h exhibited remarkable osteoinductive properties and excellent homogeneous bone formation. Our studies suggested that this novel collagen-based BMP-2 targeting bone repair system induced better bone formation not only in quantity but also in quality. Similar approaches may also be used for the repair of other tissue injuries.     

Potentiation of the activity of bone morphogenetic protein-2 in bone regeneration by a PLA-PEG/hydroxyapatite composite

Bone morphogenetic proteins (BMPs) are biologically active molecules capable of inducing new bone formation, and show potential for clinical use in bone defect repair. However, an ideal system for delivering BMPs that can potentiate their bone-inducing ability and provide initial mechanical strength and scaffold for bone ingrowth has not yet been developed. In this study, to construct a carrier/scaffold system for BMPs, we combined two biomaterials: interconnected-porous calcium hydroxyapatite ceramics (IP-CHA), and the synthetic biodegradable polymer poly D,L,-lactic acid-polyethyleneglycol block co-polymer (PLA-PEG). We used a rabbit radii model to evaluate the bone-regenerating efficacy of rhBMP-2/PLA-PEG/IP-CHA composite. At 8 weeks after implantation, all bone defects in groups treated with 5 or 20 microg of BMP were completely repaired with sufficient strength. Furthermore, using this carrier scaffold system, we reduced the amount of BMP necessary for such results to about a tenth of the amount needed in previous studies, probably due to the superior osteoconduction ability of IP-CHA and the optimal drug delivery system provided by PLA-PEG, inducing new bone formation in the interconnected pores. The present findings indicate that the synthetic biodegradable polymer/IP-CHA composite is an excellent combination carrier/scaffold delivery system for rhBMP-2, and that it strongly promotes the clinical effects of rhBMP-2 in bone tissue regeneration.     

Augmented bone regeneration activity of platelet-rich plasma by biodegradable gelatin hydrogel

This study investigates the ability of platelet-rich plasma (PRP) combined with biomaterials to enhance in vivo bone-repairing activity. A biodegradable hydrogel was prepared from gelatin, which has an affinity for various growth factors. Rabbit PRP was conventionally prepared by blood centrifugation and dropped onto freeze-dried gelatin hydrogel to obtain gelatin hydrogel incorporating PRP. Gelatin hydrogel incorporating PRP was applied to a bone defect of rabbit ulna to evaluate bone formation at the defect in terms of soft X-ray and histological examinations. As controls, fibrin incorporating PRP, empty gelatin hydrogel, and free PRP were applied to the defect; in addition, defect without any application was examined. Successful bone regeneration was observed at bone defect treated with gelatin hydrogel incorporating PRP, in marked contrast to the control groups. When in contact with gelatin, growth factors, such as platelet-derived growth factor and transforming growth factor beta(1), were released from the PRP. PRP growth factors are immobilized in the hydrogel through physicochemical interaction with gelatin molecules. The immobilized growth factors are released from the hydrogel in concert with hydrogel degradation. It is likely that the gelatin hydrogel permitted the controlled release of bioactive growth factors, resulting in factor-induced promotion of bone regeneration.     

Enhanced guided bone regeneration by controlled tetracycline release from poly(L-lactide) barrier membranes

With the aim of providing effective periodontal therapeutic modality, drug-releasing membranes for guided tissue regeneration (GTR) were developed. As GTR membranes, biodegradable barrier membranes composed of porous poly(L-lactide) (PLLA) films cast on poly(glycolide) (PGA) meshes were fabricated using an in-air drying phase inversion technique. PLLA was dissolved in methylene chloride-ethylacetate mixtures, cast on knitted PGA mesh, and then air-dried. Tetracycline, which is used in periodontal therapy because of its antibacterial activity and tissue regenerating effects, including osteoblast chemotactic effect and anti-collagenolytic activity, was incorporated into the membranes by adding it to PLLA solutions. The guided bone regenerating potential of tetracycline-loaded membranes was evaluated using release kinetics both in vitro and in vivo, biodegradation tests, and cell attachment tests. Homogeneous pores were generated both at the surface and in a sublayer of the membranes. The release kinetics of tetracycline depended mainly upon the hydrophilicity of tetracycline and the porosity of the membrane. The release rate further could be controlled by loaded drug contents. The release of tetracycline was appropriate for maintaining anti-microbial activity and for its tissue-regenerating potential. The membranes retained a proper degradation property, maintaining their mechanical integrity for the barrier function for 4 weeks. Tetracycline-loaded membranes induced increased cell attachment levels compared with those of unloaded membranes. Tetracycline-loaded membranes markedly increased new bone formation in rat calvarial defects and induced bony reunion after 2 weeks of implantation. These results suggest that tetracycline-loaded PLLA membranes potentially enhance guided tissue regenerative efficacy.     

Delivery of plasmid DNA encoding bone morphogenetic protein-2 with a biodegradable branched polycationic polymer in a critical-size rat cranial defect model

This study investigated the delivery of plasmid DNA (pDNA) encoding bone morphogenetic protein-2 in the form of polyplexes with a biodegradable branched triacrylate/amine polycationic polymer (TAPP) that were complexed with gelatin microparticles (GMPs) loaded within a porous tissue engineering scaffold. More specifically, the study investigated the interplay between TAPP degradation, gelatin degradation, pDNA release, and bone formation in a critical-size rat cranial defect model. The pDNA release kinetics in vitro were not affected by the crosslinking density of the GMPs but depended, rather, on the degradation rates of the TAPPs. Besides the initial release of polyplexes not bound to the GMPs and the minimal release of polyplexes through diffusion or dissociation from the GMPs, the pDNA was likely released as naked pDNA or as part of an incomplete polyplex, after the degradation of fragments of the polycationic polymer. After 30 days, significantly higher amounts of pDNA were released (93%-98%) from composite scaffolds containing naked pDNA or pDNA complexed with P-AEPZ (synthesized with 1-[2-aminoethyl]piperazine, a faster degrading TAPP) compared with those containing pDNA complexed with P-DED (synthesized with N,N-dimethylethylenediamine, a slower degrading TAPP) (74%-82%). Composite scaffolds containing GMPs complexed with TAPP/pDNA polyplexes did not result in enhanced bone formation, as analyzed by microcomputed tomography and histology, in a critical-size rat cranial defect at 12 weeks postimplantation compared with those loaded with naked pDNA. The results demonstrate that polycationic polymers with a slow degradation rate can prolong the release of pDNA from the composite scaffolds and suggest that a gene delivery system comprising biodegradable polycationic polymers should be designed to release the pDNA in an intact polyplex form.