Multi-functional P(3HB) microsphere/45S5 Bioglass®-based composite scaffolds for bone tissue engineering

Novel multi-functional P(3HB) microsphere/45S5 Bioglass®-based composite scaffolds exhibiting potential for drug delivery were developed for bone tissue engineering. 45S5 Bioglass®-based glass–ceramic scaffolds of high interconnected porosity produced using the foam-replication technique were coated with biodegradable microspheres (size < 2 μm) made from poly(3-hydroxybutyrate), P(3HB), produced using Bacillus cereus SPV. A solid-in-oil-in-water emulsion solvent extraction/evaporation technique was used to produce these P(3HB) microspheres. A simple slurry-dipping method, using a 1 wt.% suspension of P(3HB) microspheres in water, dispersed by an ultrasonic bath, was used to coat the scaffold, producing a uniform microsphere coating throughout the three-dimensional scaffold structure. Compressive strength tests confirmed that the microsphere coating slightly enhanced the scaffold mechanical strength. It was also confirmed that the microsphere coating did not inhibit the bioactivity of the scaffold when immersed in simulated body fluid (SBF) for up to 4 weeks. The hydroxyapatite (HA) growth rate on P(3HB) microsphere-coated 45S5 Bioglass® composite scaffolds was very similar to that on the uncoated control sample, qualitatively indicating similar bioactivity. However, the surface topography of the HA surface layer was affected as shown by results obtained from white light interferometry. The roughness of the surface was much higher for the P(3HB) microsphere-coated scaffolds than for the uncoated samples, after 7 days in SBF. This feature would facilitate cell attachment and proliferation. Finally, gentamycin was successfully encapsulated into the P(3HB) microspheres to demonstrate the drug delivery capability of the scaffolds. Gentamycin release kinetics was determined using liquid chromatography–mass spectrometry. The release of the drug from the coated composite scaffolds was slow and controlled when compared to the observed fast and relatively uncontrolled drug release from the bone scaffold (without microsphere coating). Thus, this unique multifunctional bioactive composite scaffold has the potential to enhance cell attachment and to provide controlled delivery of relevant drugs for bone tissue engineering.     

Effect of local sequential VEGF and BMP-2 delivery on ectopic and orthotopic bone regeneration

Bone regeneration is a coordinated cascade of events regulated by several cytokines and growth factors. Angiogenic growth factors are predominantly expressed during the early phases for re-establishment of the vascularity, whereas osteogenic growth factors are continuously expressed during bone formation and remodeling. Since vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs) are key regulators of angiogenesis and osteogenesis during bone regeneration, the aim of this study was to investigate if their sequential release could enhance BMP-2-induced bone formation. A composite consisting of poly(lactic-co-glycolic acid) microspheres loaded with BMP-2 embedded in a poly(propylene) scaffold surrounded by a gelatin hydrogel loaded with VEGF was used for the sequential release of the growth factors. Empty composites or composites loaded with VEGF and/or BMP-2 were implanted ectopically and orthotopically in Sprague–Dawley rats (n = 9). Following implantation, the local release profiles were determined by measuring the activity of ¹²⁵I-labeled growth factors using scintillation probes. After 8 weeks blood vessel and bone formation were analyzed using microangiography, μCT and histology. The scaffolds exhibited a large initial burst release of VEGF within the first 3 days and a sustained release of BMP-2 over the full 56-day implantation period. Although VEGF did not induce bone formation, it did increase the formation of the supportive vascular network (p = 0.03) in ectopic implants. In combination with local sustained BMP-2 release, VEGF significantly enhanced ectopic bone formation compared to BMP-2 alone (p = 0.008). In the orthotopic defects, no effect of VEGF on vascularisation was found, nor was bone formation higher by the combination of growth factors, compared to BMP-2 alone. This study demonstrates that a sequential angiogenic and osteogenic growth factor release may be beneficial for the enhancement of bone regeneration.     

Fabrication and characterization of monodisperse PLGA–alginate core–shell microspheres with monodisperse size and homogeneous shells for controlled drug release

Monodisperse PLGA–alginate core–shell microspheres with controlled size and homogeneous shells were first fabricated using capillary microfluidic devices for the purpose of controlling drug release kinetics. Sizes of PLGA cores were readily controlled by the geometries of microfluidic devices and the fluid flow rates. PLGA microspheres with sizes ranging from 15 to 50 μm were fabricated to investigate the influence of the core size on the release kinetics. Rifampicin was loaded into both monodisperse PLGA microspheres and PLGA–alginate core–shell microspheres as a model drug for the release kinetics studies. The in vitro release of rifampicin showed that the PLGA core of all sizes exhibited sigmoid release patterns, although smaller PLGA cores had a higher release rate and a shorter lag phase. The shell could modulate the drug release kinetics as a buffer layer and a near-zero-order release pattern was observed when the drug release rate of the PLGA core was high enough. The biocompatibility of PLGA–alginate core–shell microspheres was assessed by MTT assay on L929 mouse fibroblasts cell line and no obvious cytotoxicity was found. This technique provides a convenient method to control the drug release kinetics of the PLGA microsphere by delicately controlling the microstructures. The obtained monodisperse PLGA–alginate core–shell microspheres with monodisperse size and homogeneous shells could be a promising device for controlled drug release.     

Therapeutic bioactive microcarriers: Co-delivery of growth factors and stem cells for bone tissue engineering

Novel microcarriers made of sol–gel-derived bioactive glasses were developed for delivering therapeutic molecules effectively while cultivating stem cells for bone tissue engineering. Silica sols with varying concentration of Ca (0–30 mol.%) were formulated into microspheres ranging from 200 to 300 μm under optimized conditions. A highly mesoporous structure was created, with mesopore sizes of 2.5–6.3 nm and specific surface areas of 420–710 m² g⁻¹, which was highly dependent on the Ca concentration. Therapeutic molecules could be effectively loaded within the mesoporous microcarriers during microsphere formulation. Cytochrome C (cyt C), used as a model protein for the release study, was released in a highly sustainable manner, with an almost zero-order kinetics over a period of months; the amount released was ∼2% at 9 days, and 15% at 40 days. A slight increase in the release rate was observed in the microcarrier containing Ca, which was related to the dissolution rate and pore size. The presence of Ca accelerated the formation of hydroxyapatite on the surface of the microcarriers. Cells cultured on the bioactive microcarriers were well adhered and distributed, and proliferated actively, confirming the three-dimensional substrate role of the microcarriers. An in vivo study performed in a rat subcutaneous model demonstrated the satisfactory biocompatibility of the prepared microspheres. As a therapeutic target molecule, basic fibroblast growth factor (bFGF) was incorporated into the microcarriers. A slow release pattern similar to that of cyt C was observed for bFGF. Cells adhered and proliferated to significantly higher levels on the bFGF-loaded microcarriers, demonstrating the effective role of bFGF in cell proliferative potential. It is believed that the developed mesoporous bioactive glass microspheres represent a new class of therapeutic cell delivery carrier, potentially useful in the sustainable delivery of therapeutic molecules such as growth factors, as well as in the support of stem cell proliferation and osteogenesis for bone tissue engineering.     

Dual pH- and temperature-responsive microparticles for protein delivery to ischemic tissues

Injectable “smart” microspheres that are sensitive to both temperature and pH have been fabricated and tested for controlled delivery of therapeutic proteins to ischemic skeletal muscle. A library of copolymers composed of N-isopropyl acrylamide (NIPAAm), propyl acrylic acid (PAA), and butyl acrylate (BA) was used to fabricate microspheres using a double emulsion method, and an optimal formulation made from copolymers composed of 57 mol.% NIPAAm, 18 mol.% PAA and 25 mol.% BA copolymers was identified. At 37 °C and pH representative of ischemic muscle (i.e. pH 5.2–7.2), these microspheres produced sustained, diffusion-controlled release, and at normal, physiological pH (i.e. pH 7.4), they underwent dissolution and rapid clearance. Delivery of fibroblast growth factor 2 was used to confirm that protein bioactivity was retained following microsphere encapsulation/release based on a dose-dependent increase in NIH3T3 fibroblast proliferation in vitro. Microsphere-loaded or free Cy5.5-labeled albumin was injected into ischemic and control gastrocnemii of mice following unilateral induction of hind limb ischemia to model peripheral arterial disease. In the ischemic limb at days 3.5 and 7, there was higher local retention of the protein delivered via microspheres relative to injected free protein (p < 0.05). However, clearance of protein delivered via microspheres was equivalent to free protein at later time points that correspond to ischemic recovery in this model. Finally, histological analysis of the gastrocnemius revealed that the polymeric microspheres did not produce any microscopic signs of toxicity near the injection site. These combined results suggest that the pH- and temperature-responsive microspheres presented herein are a promising technological platform for controlled protein delivery to ischemic tissue.     

Poly(l-glutamic acid)/chitosan polyelectrolyte complex porous microspheres as cell microcarriers for cartilage regeneration

In this study a novel kind of porous poly(l-glutamic acid) (PLGA)/chitosan polyelectrolyte complex (PEC) microsphere was developed through electrostatic interaction between PLGA and chitosan. By adjusting the formula parameters chitosan microspheres with an average pore size of 47.5 ± 5.4 μm were first developed at a concentration of 2 wt.% and freeze temperature of −20 °C. For self-assembly of the PEC microspheres porous chitosan microspheres were then incubated in PLGA solution at 37 °C. Due to electrostatic interaction a large amount of PLGA (110.3 μg mg⁻¹) was homogeneously absorbed within the chitosan microspheres. The developed PEC microspheres retained their original size, pore diameters and interconnected porous structure. Fourier transform infrared spectroscopy, thermal gravimetric analysis and zeta potential analysis revealed that the PEC microspheres were successfully prepared through electrostatic interaction. Compared with microspheres fabricated from chitosan, the porous PEC microspheres were shown to efficiently promote chondrocyte attachment and proliferation. After injection subcutaneously for 8 weeks PEC microspheres loaded with chondrocytes were found to produce significant more cartilaginous matrix than chitosan microspheres. These results indicate that these novel fabricated porous PLGA/chitosan PEC microspheres could be used as injectable cell carriers for cartilage tissue engineering.     

VEGF-releasing suture material for enhancement of vascularization: Development,in vitro and in vivo study

As it has been demonstrated that bioactive substances can be delivered locally using coated surgical suture materials, the authors developed a vascular endothelial growth factor (VEGF)-releasing suture material that should promote vascularization and potentially wound healing. In this context, the study focused on the characterization of the developed suture material and the verification of its biological activity, as well as establishing a coating process that allows reproducible and stable coating of a commercially available polydioxanone suture material with poly(l-lactide) (PLLA) and 0.1 μg and 1.0 μg VEGF. The in vitro VEGF release kinetics was studied using a Sandwich ELISA. The biological activity of the released VEGF was investigated in vitro using human umbilical vein endothelial cells. The potential of the VEGF-releasing suture material was also studied in vivo 5 days after implantation in the hind limb of Wistar rats, when the histological findings were analyzed. The essential results, enhanced cell viability in vitro as well as significantly increased vascularization in vivo, were achieved using PLLA/1.0 μg VEGF-coated suture material. Furthermore, ELISA measurements revealed a high reproducibility of the VEGF release behavior. Based on the results achieved regarding the dose–effect relationship of VEGF, the stability during its processing and the release behavior, it can be predicted that a bioactive suture material would be successful in later in vivo studies. Therefore, this knowledge could be the basis for future studies, where bioactive substances with different modes of action are combined for targeted, overall enhancement of wound healing.     

Controlled release of vascular endothelial growth factor using poly-lactic-co-glycolic acid microspheres: in vitro characterization and application in polycaprolactone fumarate nerve conduits

Vascular endothelial growth factor (VEGF) is a potent angiogenic stimulator. Controlled release of such stimulators may enhance and guide the vascularization process, and when applied in a nerve conduit may play a role in nerve regeneration. We report the fabrication and in vitro characterization of poly-lactic-co-glycolic acid (PLGA) microspheres encapsulating VEGF and the in vivo application of nerve conduits supplemented with VEGF-containing microspheres. PLGA microspheres containing VEGF were prepared by the double emulsion-solvent evaporation technique. This yielded 83.16% of microspheres with a diameter <53 μm. VEGF content measured by ELISA indicated 93.79±10.64% encapsulation efficiency. Release kinetics were characterized by an initial burst release of 67.6±8.25% within the first 24h, followed by consistent release of approximately 0.34% per day for 4 weeks. Bioactivity of the released VEGF was tested by human umbilical vein endothelial cell (HUVEC) proliferation assay. VEGF released at all time points enhanced HUVEC proliferation, confirming that VEGF retained its bioactivity throughout the 4 week time period. When the microsphere delivery system was placed in a biosynthetic nerve scaffold robust nerve regeneration was observed. This study established a novel system for controlled release of growth factors and enables in vivo studies of nerve conduits conditioned with this system.     

An in situ forming biodegradable hydrogel-based embolic agent for interventional therapies

We present here the characteristics of an in situ forming hydrogel prepared from carboxymethyl chitosan and oxidized carboxymethyl cellulose for interventional therapies. Gelation, owing to the formation of Schiff bases, occurred both with and without the presence of a radiographic contrast agent. The hydrogel exhibited a highly porous internal structure (pore diameter 17 ± 4 μm), no cytotoxicity to human umbilical vein endothelial cells, hemocompatibility with human blood, and degradability in lysozyme solutions. Drug release from hydrogels loaded with a sclerosant, tetracycline, was measured at pH 7.4, 6 and 2 at 37 °C. The results showed that tetracycline was more stable under acidic conditions, with a lower release rate observed at pH 6. An anticancer drug, doxorubicin, was loaded into the hydrogel and a cumulative release of 30% was observed over 78 h in phosphate-buffered saline at 37 °C. Injection of the hydrogel precursor through a 5-F catheter into a fusiform aneurysm model was feasible, leading to complete filling of the aneurysmal sac, which was visualized by fluoroscopy. The levels of occlusion by hydrogel precursors (1.8% and 2.1%) and calibrated microspheres (100–300 μm) in a rabbit renal model were compared. Embolization with hydrogel precursors was performed without clogging and the hydrogel achieved effective occlusion in more distal arteries than calibrated microspheres. In conclusion, this hydrogel possesses promising characteristics potentially beneficial for a wide range of vascular intervention procedures that involve embolization and drug delivery.     

In vitro and in vivo evaluation of biodegradable embolic microspheres with tunable anticancer drug release

Natural polymer-derived materials have attracted increasing interest in the biomedical field. Polysaccharides have obvious advantages over other polymers employed for biomedical applications due to their exceptional biocompatibility and biodegradability. None of the spherical embolic agents used clinically is biodegradable. In the current study, microspheres prepared from chitosan and carboxymethyl cellulose (CMC) were investigated as a biodegradable embolic agent for arterial embolization applications. Aside from the enzymatic degradability of chitosan units, the cross-linking bonds in the matrix, Schiff bases, are susceptible to hydrolytic cleavage in aqueous conditions, which would overcome the possible shortage of enzymes inside the arteries. The size distribution, morphology, water retention capacity and degradability of the microspheres were found to be affected by the modification degree of CMC. An anticancer drug, doxorubicin, was successfully incorporated into these microspheres for local release and thus for killing cancerous cells. These microspheres demonstrated controllable degradation time, variable swelling and tunable drug release profiles. Co-culture with human umbilical vein endothelial cells revealed non-cytotoxic nature of these microspheres compared to monolayer control (P > 0.95). In addition, a preliminary study on the in vivo degradation of the microspheres (100–300 μm) was performed in a rabbit renal embolization model, which demonstrated that the microspheres were compatible with microcatheters for delivery, capable of occluding the arteries, and biodegradable inside arteries. These microspheres with biodegradability would be promising for embolization therapies.     

Development and in vitro characterization of vascular endothelial growth factor (VEGF)-loaded poly(DL-lactic-co-glycolic acid)/poly(ethylene glycol) microspheres using a solid encapsulation/single emulsion/solvent extraction technique

Poly(DL-lactide-co-glycolide) (PLGA)/polyethylene glycol (PEG) microspheres are one modality of controlled delivery of biologically active molecules that would further the development of engineered tissues. As a possible mechanism to stimulate angiogenesis within an engineered tissue, vascular endothelial growth factor (VEGF) and bovine serum albumin (BSA) were coencapsulated into microspheres fabricated from PEG and 50/50 PLGA using a solid-encapsulation/single-emulsion/solvent extraction technique. Two VEGF/BSA ratios were studied: 1:2000 and 1:10,000. Analysis consisted of the loading efficiency, particle size distribution, bright-field microscopy, scanning electron microscopy, release kinetics, and an in vitro human umbilical vein endothelial cell proliferation assay to assess biological activity of the released VEGF. Results show the microspheres could be manufactured, stored, and degraded over 28 days. The burst release rates for 1:2000 and 1:10,000 VEGF/BSA microspheres were 71.87 +/- 8.11 and 27.91 +/- 1.71 ng/mL (mean +/- standard error of the mean), respectively; steady-state release rates were 6.56 +/- 1.10 and 2.21 +/- 0.47 ng/mL, respectively. The microspheres released biologically active VEGF, and the VEGF increased the proliferation of HUVECs in culture (p <.05). The successful development of a novel, cost-effective, scalable technique for producing microspheres loaded with biologically active proteins is presented. Using the data obtained from these studies, a defined concentration of microspheres will deliver a quantifiable level of VEGF at a known release rate.     

The effect of dual frequency cyclic compression on matrix deposition by osteoblast-like cells grown in 3D scaffolds and on modulation of VEGF variant expression

As a strategy to optimise osteointegration of biomaterials by inducing proper extracellular matrix synthesis, and specifically angiogenic growth factor production and storage, we tested the effects of cyclic mechanical compression on 3D cultures of human osteoblast-like cells. MG-63 cells were seeded into 3D porous hydroxyapatite ceramics under vacuum to enable a homogenous cellular distribution. A four-day culture period allowed cell proliferation throughout the scaffolds. Low amplitude cyclic compressions were then applied to the scaffolds for 15 min with different regimens generated by the ZetOS? system. A 3 Hz sinusoidal (sine) signal increased slightly collagen and fibronectin expression. When 50 Hz or 100 Hz vibrations were superimposed to the 3 Hz signal, matrix protein expression was down-regulated. In contrast, adding a 25 Hz vibration up-regulated significantly collagen and fibronectin. Moreover, expression of a matrix-bound variant of vascular endothelial growth factor-A (VEGF-A) was specifically stimulated compared to control or 3 Hz sine, and non-soluble VEGF protein was increased. Our study enabled us to identify low-amplitude, high-frequency strain regimen able to increase major matrix proteins of bone tissue and to regulate the expression of VEGF variants, showing that an appropriate combined loading has the potential to functionalise cellularized bone-like constructs.     

The in vivo performance of CaP/PLGA composites with varied PLGA microsphere sizes and inorganic compositions

Enrichment of calcium phosphate (CaP) bone substitutes with poly(lactic-co-glycolic acid) (PLGA) microspheres to create porosity overcomes the problem of poor CaP degradation. The degradation of CaP–PLGA composites can be customized by changing the physical and chemical properties of PLGA and/or CaP. However, the effect of the size of dense (solid rather than hollow) PLGA microspheres in CaP has not previously been described. The present study aimed at determining the effect of different dense (i.e. solid) PLGA microsphere sizes (small (S) ～20 μm vs. large (L) ～130 μm) and of CaP composition (CaP with either anhydrous dicalcium phosphate (DCP) or calcium sulphate dihydrate (CSD)) on CaP scaffold biodegradability and subsequent bone in-growth. To this end mandibular defects in minipigs were filled with pre-set CaP–PLGA implants, with autologous bone being used as a control. After 4 weeks the autologous bone group outperformed all CaP–PLGA groups in terms of the amount of bone present at the defect site. On the other hand, at 12 weeks substantial bone formation was observed for all CaP–PLGA groups (ranging from 47 ± 25% to 62 ± 15%), showing equal amounts of bone compared with the autologous bone group (82 ± 9%), except for CaP with DCP and large PLGA microspheres (47 ± 25%). It was concluded that in the current study design the difference in PLGA microsphere size and CaP composition led to similar results with respect to scaffold degradation and subsequent bone in-growth. Further, after 12 weeks all CaP–PLGA composites proved to be effective for bone substitution.     

Cryopreservable and tumorigenic three-dimensional tumor culture in porous poly(lactic-co-glycolic acid) microsphere

In vitro tumor models that mimic in vivo conditions may be ideal for screening anticancer drugs and their formulations and developing tumors in animal models. Three-dimensional (3-D) culture of cancer cells on polymeric scaffolds can be an option for such models. In the present study, porous poly(lactic acid-co-glycolic acid) (PLGA) microsphere was used both as a cancer cell culture substrate to expand cells and as a cancer cell transplantation vehicle for tumor construction in mice. MCF-7 cells cultured on porous PLGA microspheres in stirred suspension bioreactors expanded by 2.8-fold over seven days and maintained viability. At three months after inoculation with 2 × 10⁶ cells/site, the tumor formation by MCF-7 cells cultured on microspheres was much more effective (4 tumors/5 mice) than its counterpart cultured on plates (1/5). More importantly, cell viability and metabolic activity were not significantly changed even after one freeze–thaw cycle of the 3-D culture. MCF-7 cells cultured on the microspheres and the cells in 3-D after cryopreservation were more resistant to doxorubicin than MCF-7 cells cultured on plates.     

Engineering surfaces for site-specific vascular differentiation of mouse embryonic stem cells

Differentiation of stem and progenitor cells routinely relies on the application of soluble growth factors, an approach that enables temporal control of cell fate but enables no spatial control of the differentiation process. Angiogenic progenitor cells derived from mouse embryonic stem cells (ESCs) were differentiated here according to the pattern of immobilized vascular endothelial growth factor-A (VEGF). Mouse ESCs engineered to express green fluorescent protein (eGFP) under control of promoter for the receptor tyrosine kinase Flk1 were used. The Flk1+ angiogenic progenitors were selected from day 3 differentiating embryoid bodies based on their expression of eGFP using fluorescence activated cell sorting. Mouse VEGF₁₆₅ was covalently immobilized onto collagen IV (ColIV) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) chemistry. A non-cell adhesive layer of photocrosslinkable chitosan was first created, after which VEGF–ColIV was stamped as 100 μm wide lanes on top of the chitosan layer and the Flk1+ angiogenic progenitors were seeded for site-specific differentiation. Lanes stamped with only ColIV served as controls. The results presented here demonstrate that the cultivation of Flk1+ progenitors on surfaces with immobilized VEGF yielded primarily endothelial cells (53 ± 13% CD31 positive and 17 ± 2% smooth muscle actin positive), whereas surfaces without VEGF favored vascular smooth muscle-like cell differentiation (26 ± 17% CD31 positive and 38 ± 9% smooth muscle actin positive).     

In vivo degradation of calcium phosphate cement incorporated into biodegradable microspheres

In this study we have investigated the influence of the mechanism of microsphere degradation or erosion on the in vivo degradation of microsphere/calcium phosphate cement composites (microsphere CPCs) used in tissue engineering. Microspheres composed of poly(lactic-co-glycolic acid) (PLGA), gelatin and poly(trimethylene carbonate) (PTMC) were used as the model and the resulting microsphere CPCs were implanted subcutaneously for 4, 8 or 12 weeks in the back of New Zealand white rabbits. Besides degradation, the soft tissue response to these formulations was evaluated. After retrieval, specimens were analyzed by physicochemical characterization and histological analysis. The results showed that all microsphere CPCs exhibited microsphere degradation after 12 weeks of subcutaneous implantation, which was accompanied by decreasing compression strength. The PLGA microspheres exhibited bulk erosion simultaneously throughout the whole composite, whereas the gelatin type B microspheres were degradated from the outside to the center of the composite. High molecular weight PTMC microspheres exhibited surface erosion resulting in decreasing microsphere size. Furthermore, all composites showed a similar tissue response, with decreasing capsule thickness over time and a persistent moderate inflammatory response at the implant interface. In conclusion, microsphere CPCs can be used to generate porous scaffolds in an in vivo environment after degradation of microspheres by various degradation/erosion mechanisms.     

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.     

In vitro characterization of vascular endothelial growth factor and dexamethasone releasing hydrogels for implantable probe coatings

Anti-fouling hydrogel coatings, copolymers of 2-hydroxyethyl methacrylate, 1-vinyl-2-pyrrolidinone, and polyethylene glycol, were investigated for the purpose of improving biosensor biocompatibility. These coatings were modified to incorporate poly(lactide-co-glycolide) (PLGA) microspheres in order to release dexamethasone (DX) and/or vascular endothelial growth factor (VEGF). DX and VEGF release kinetics from microspheres, hydrogels, and microspheres embedded in hydrogels were determined in 2-week and 1-month studies. Overall, monolithic, non-degradable hydrogel drug release had an initial burst followed by release at a significantly lower amount. Microsphere drug release kinetics exhibited an initial burst followed by sustained release for 1 month. Embedding microspheres in hydrogels resulted in attenuated drug delivery. VEGF release from embedded microspheres, 1.1+/-0.3 ng, was negligible compared to release from hydrogels, 197+/-33 ng. After the initial burst from DX-loaded hydrogels, DX release from embedded microspheres was similar to that of hydrogels. The total DX release from hydrogels, 155+/-35 microg, was greater than that of embedded microspheres, 60+/-6 microg. From this study, hydrogel sensor coatings should be prepared incorporating VEGF in the hydrogel and DX either in the hydrogel or in DX microspheres embedded in the hydrogel.     

Transfection of macrophages by collagen hollow spheres loaded with polyplexes: A step towards modulating inflammation

Macrophages are key orchestrators of inflammation as they secrete proteases and inflammatory cytokines. To date, therapies aimed at modulating macrophage phenotype have failed due to the short half-life of biomolecules in the body. Therefore, inhibition of inflammation by gene therapy constitutes a new hope.In the present study, we have assessed collagen hollow spheres as a reservoir system for polyplexes in order to transfect human macrophages while preserving cell viability. Polyplexes were formed by complexing G-Luc plasmid with a poly(2-dimethylaminoethyl methacrylate) poly(ethylene glycol) based hyperbranched polymer. Several ratios of polymer/pDNA (5:1, 8:1, 10:1 w/w) complexes in two different sphere sizes (1.24 and 4.5 μm) were tested. Collagen hollow spheres were loaded with polyplexes up to 80 μg of pDNA per mg of microspheres. The release of polyplexes from the spheres was delayed and prolonged i.e. 20% of the initial amount released in 5 days. Following incubation with polyplex-loaded microspheres, macrophages were transfected (polyplex pDNA:polymer ratio 1:10 w/w). In addition, collagen hollow spheres maintained cell viability as more than 80% of cells were viable after 4 days in culture. In contrast, when used alone, polyplexes were seen to be toxic, while there was no transfection detected. Taken together, these results show that collagen hollow spheres may be used as a reservoir for controlled gene delivery to macrophages. Unlike existing gene delivery systems, this system allows for macrophage transfection with minimal toxicity. Hence, this system has a potential for the delivery of a therapeutic gene in order to modulate inflammation.     

Injectable and porous PLGA microspheres that form highly porous scaffolds at body temperature

Injectable scaffolds are of interest in the field of regenerative medicine because of their minimally invasive mode of delivery. For tissue repair applications, it is essential that such scaffolds have the mechanical properties, porosity and pore diameter to support the formation of new tissue. In the current study, porous poly(dl-lactic acid-co-glycolic acid) (PLGA) microspheres were fabricated with an average size of 84 ± 24 μm for use as injectable cell carriers. Treatment with ethanolic sodium hydroxide for 2 min was observed to increase surface porosity without causing the microsphere structure to disintegrate. This surface treatment also enabled the microspheres to fuse together at 37 °C to form scaffold structures. The average compressive strength of the scaffolds after 24 h at 37 °C was 0.9 ± 0.1 MPa, and the average Young’s modulus was 9.4 ± 1.2 MPa. Scaffold porosity levels were 81.6% on average, with a mean pore diameter of 54 ± 38 μm. This study demonstrates a method for fabricating porous PLGA microspheres that form solid porous scaffolds at body temperature, creating an injectable system capable of supporting NIH-3T3 cell attachment and proliferation in vitro.