RhBMP-2 microspheres-loaded chitosan/collagen scaffold enhanced osseointegration: an experiment in dog

The purpose of this study is to develop a novel recombinant human bone morphogenetic protein-2 (rhBMP-2) sustained release scaffold for dental implant osseointegration, and to evaluate the effect of this scaffold on promoting bone formation. RhBMP-2 was encapsulated in the poly-D,L-lactide-co-glycolide (PLGA) biodegradable microspheres, which were subsequently dispersed in a chitosan/collagen composite scaffold. This rhBMP-2 microspheres-loaded scaffold (S-MB) was compared with a chitosan/collagen scaffold without microspheres that directly encapsulated rhBMP-2 (S-B) in vitro and in vivo. The microstructure of the new scaffold was examined with scanning electron microscopy. The release profile of rhBMP-2 in vitro was measured at interval periods. The effect of rhBMP-2 encapsulated scaffolds on enhancing bone formation through implantation in dogs' mandibles was identified by histological examination of the regenerated bone after 4 weeks of implantation. Due to PLGA microspheres being loaded, the S-MB exhibited lower values at porosity and swelling rate, as well as a higher effective release dose than that of the S-B. Bone density, bone-implant contact, and bone-fill values measured from dog experiments demonstrated that the S-MB induced bone regeneration more quickly and was timely substituted by new bone. It was concluded that this sustained carrier scaffold based on microspheres was more effective to induce implant osseointegration.     

A dual-application poly (dl-lactic-co-glycolic) acid (PLGA)-chitosan composite scaffold for potential use in bone tissue engineering

The development of patient-friendly alternatives to bone-graft procedures is the driving force for new frontiers in bone tissue engineering. Poly (dl-lactic-co-glycolic acid) (PLGA) and chitosan are well-studied and easy-to-process polymers from which scaffolds can be fabricated. In this study, a novel dual-application scaffold system was formulated from porous PLGA and protein-loaded PLGA/chitosan microspheres. Physicochemical and in vitro protein release attributes were established. The therapeutic relevance, cytocompatibility with primary human mesenchymal stem cells (hMSCs) and osteogenic properties were tested. There was a significant reduction in burst release from the composite PLGA/chitosan microspheres compared with PLGA alone. Scaffolds sintered from porous microspheres at 37 °C were significantly stronger than the PLGA control, with compressive strengths of 0.846 ± 0.272 MPa and 0.406 ± 0.265 MPa, respectively (p < 0.05). The formulation also sintered at 37 °C following injection through a needle, demonstrating its injectable potential. The scaffolds demonstrated cytocompatibility, with increased cell numbers observed over an 8-day study period. Von Kossa and immunostaining of the hMSC-scaffolds confirmed their osteogenic potential with the ability to sinter at 37 °C in situ.     

Apatite-coated poly(lactic-co-glycolic acid) microspheres as an injectable scaffold for bone tissue engineering

Biodegradable polymer/ceramic composite scaffold could overcome limitations of biodegradable polymers or ceramics for bone regeneration. Injectable scaffold has raised great interest for bone regeneration in vivo, since it allows one for easy filling of irregularly shaped bone defects and implantation of osteogenic cells through minimally invasive surgical procedures The purpose of this study was to determine whether apatite-coated poly(lactic-co-glycolic acid) (PLGA) microspheres could be used as an injectable scaffold to regenerate bone in vivo. Apatite-coated PLGA microspheres were fabricated by incubating PLGA microspheres in simulated body fluid. The apatite that coated the PLGA microsphere surfaces was similar to apatite in natural bone, as demonstrated by scanning electron microscopy, X-ray diffraction spectra, energy-dispersive spectroscopy, and Fourier transformed-infrared spectroscopy analyses. Rat osteoblasts were mixed with apatite-coated PLGA microspheres and injected immediately into subcutaneous sites of athymic mice. Osteoblast transplantation with plain PLGA microspheres served as a control. Histological analysis of the implants at 6 weeks with hematoxylin and eosin staining, Masson's trichrome staining, and von Kossa staining revealed much better regeneration of bone in the apatite-coated PLGA microsphere group than the plain PLGA microsphere group. The new bone formation area and the calcium content of the implants were significantly higher in the apatite-coated PLGA microsphere group than in the plain PLGA microsphere group. This study demonstrates the feasibility of using apatite-coated PLGA microspheres as an injectable scaffold for in vivo bone tissue engineering. This scaffold may be useful for bone regeneration through minimally invasive surgical procedures in orthopedic applications.     

Generation of bioactive nano-composite scaffold of nanobioglass/silk fibroin/carboxymethyl cellulose for bone tissue engineering

Abstract

The development of bone tissue construct through tissue engineering approach offers a great promise in meeting the increasing demand for repair and regeneration of damaged and/or diseased bone tissue. For the generation of bone tissue engineered construct, polymer-ceramic composite matrices with nanostructure architecture and mesenchymal stem cells (hMSCs) of human origin are of prime requirement. Keeping these in view, in the present work a novel electrospun nanofibrous silk fibroin (SF)/carboxymethyl cellulose (CMC)/nano-bioglass (nBG) composite scaffold that mimics native bone extracellular matrix with appropriate composition was designed and fabricated by free liquid surface electrospinning technique. The scaffold possesses desired morphological, structural, biodegradability, bioactivity, surface roughness and mechanical properties thereby exhibited an excellent platform to support the growth of cells. The in-vitro culture of hMSCs over the developed scaffold has shown adhesion, proliferation and viability of cells, thus facilitated cell-scaffold construct generation and further extracellular bone matrix formation through osteogenic differentiation as evident from alkaline phosphatase activity, biomineralization, immunostaining and Runx2/osteocalcin expression assessment. Thus, the developed hMSCs seeded scaffold construct might be suitable for bone tissue engineering applications.     

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.     

Novel mesoporous silica-based antibiotic releasing scaffold for bone repair

Tissue engineering scaffolds with a micro- or nanoporous structure and able to deliver special drugs have already been confirmed to be effective in bone repair. In this paper, we first evaluated the biomineralization properties and drug release properties of a novel mesoporous silica–hydroxyapatite composite material (HMS–HA) which was used as drug vehicle and filler for polymer matrices. Biomineralization can offer a credible prediction of bioactivity for the synthetic bone regeneration materials. We found HMS–HA exhibited good apatite deposition properties after being soaked in simulated body fluid (SBF) for 7 days. Drug delivery from HMS–HA particle was in line with Fick’s law, and the release process lasted 12 h after an initial burst release with 60% drug release. A novel tissue engineering scaffold with the function of controlled drug delivery was developed, which was based on HMS–HA particles, poly(lactide-co-glycolide) (PLGA) and microspheres sintering techniques. Mechanical testing on compression, degradation behavior, pH-compensation effect and drug delivery behavior of PLGA/HMS–HA microspheres sintered scaffolds were analyzed. Cell toxicity and cell proliferation on the scaffolds was also evaluated. The results indicated that the PLGA/HMS–HA scaffolds could effectively compensate the increased pH values caused by the acidic degradation product of PLGA. The compressive strength and modulus of PLGA/HMS–HA scaffolds were remarkably high compared to pure PLGA scaffold. Drug delivery testing of the PLGA/HMS–HA scaffolds indicated that PLGA slowed gentamycin sulfate (GS) release from HMS–HA particles, and the release lasted for nearly one month. Adding HMS–HA to PLGA scaffolds improved cytocompatibility. The scaffolds demonstrated low cytotoxicity, and supported mesenchymal stem cells growth more effectively than pure PLGA scaffolds. To summarize, the data supports the development of PLGA/HMS–HA scaffolds as potential degradable and drug delivery materials for bone replacement.     

In vitro characterization of hepatocyte growth factor release from PHBV/PLGA microsphere scaffold

Polymer scaffolds which can support cells to grow as well as deliver growth factors to the cells simultaneously have great potential for the successful regeneration of failed tissues. As popularly used vehicles to deliver anti-cancer drugs and growth factors, microspheres also show many advantages as substrates to guide the growth of cells. Therefore, we aimed to examine the feasibility of using microspheres as ideal scaffolds for liver tissue engineering. To determine the capabilities of previously used microsphere scaffold to deliver growth factors simultaneously, this work investigated a long-term (about three months) release of bovine serum albumin (BSA) from microsphere scaffolds fabricated by using two different polymers, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV, 8% PHV), poly(lactide-co-glycolide) acid (PLGA, 5050) and a blend of PLGA and PHBV. BSA served as a model for hepatocyte growth factor (HGF) since both proteins have similar molecular weights and hydrophilicity. Furthermore, HGF was encapsulated into the PLGA/PHBV composite microsphere with a core-shell structure, and sustained delivery of HGF with maintained bioactivity was achieved for at least 40 days. The moderate degradation rate (about 55% loss of the initial mass) and well-preserved structure after three months of incubation indicated that the PLGA/PHBV composite microspheres would therefore be more suitable than the pure PHBV or PLGA microspheres as a scaffold for engineering liver tissue.     

Controlled drug release from a novel injectable biodegradable microsphere/scaffold composite based on poly(propylene fumarate)

The ideal biomaterial for the repair of bone defects is expected to have good mechanical properties, be fabricated easily into a desired shape, support cell attachment, allow controlled release of bioactive factors to induce bone formation, and biodegrade into nontoxic products to permit natural bone formation and remodeling. The synthetic polymer poly(propylene fumarate) (PPF) holds great promise as such a biomaterial. In previous work we developed poly(DL-lactic-co-glycolic acid) (PLGA) and PPF microspheres for the controlled delivery of bioactive molecules. This study presents an approach to incorporate these microspheres into an injectable, porous PPF scaffold. Model drug Texas red dextran (TRD) was encapsulated into biodegradable PLGA and PPF microspheres at 2 microg/mg microsphere. Five porous composite formulations were fabricated via a gas foaming technique by combining the injectable PPF paste with the PLGA or PPF microspheres at 100 or 250 mg microsphere per composite formulation, or a control aqueous TRD solution (200 microg per composite). All scaffolds had an interconnected pore network with an average porosity of 64.8 +/- 3.6%. The presence of microspheres in the composite scaffolds was confirmed by scanning electron microscopy and confocal microscopy. The composite scaffolds exhibited a sustained release of the model drug for at least 28 days and had minimal burst release during the initial phase of release, as compared to drug release from microspheres alone. The compressive moduli of the scaffolds were between 2.4 and 26.2 MPa after fabrication, and between 14.9 and 62.8 MPa after 28 days in PBS. The scaffolds containing PPF microspheres exhibited a significantly higher initial compressive modulus than those containing PLGA microspheres. Increasing the amount of microspheres in the composites was found to significantly decrease the initial compressive modulus. The novel injectable PPF-based microsphere/scaffold composites developed in this study are promising to serve as vehicles for controlled drug delivery for bone tissue engineering.     

Mechanical properties and dual drug delivery application of poly(lactic-co-glycolic acid) scaffolds fabricated with a poly(β-amino ester) porogen

Polymeric scaffolds that are biocompatible and biodegradable are widely used for tissue engineering applications. Scaffolds can be further enhanced by enabling the release of one or more drugs to stimulate regeneration or for the treatment of a specific disease or condition. In this study, poly(lactic-co-glycolic acid) (PLGA) microspheres were mixed with poly(β-amino ester) (PBAE) particles to create novel hybrid scaffolds capable of dual release of drug and growth factor. Fast-degrading PBAE particles loaded with the drug ketoprofen acted as porogens that provided a rapid 12 h release. The PLGA microspheres were loaded with a growth factor, bone morphogenetic protein 2, and fused together around the porogens to create a slow-degrading matrix that provided sustained release lasting 70 days. Drug release was further tailored by varying the amount of porogen added to the scaffold. Bioactivity measurements demonstrated that the scaffold fabrication technique did not damage the drug or protein. The compressive modulus was affected by the amount of porogen added, extending from 50 to 111 MPa for loadings from 60 to 40% PBAE, and after 5 days of degradation, it decreased to 0.6 to 1.1 kPa when the porogen was gone. PLGA containing a quick-degrading porogen can be used to release two drugs while developing a porous microarchitecture for cell ingrowth with in a matrix capable of maintaining a compressive modulus applicable for soft tissue implants.     

In vivo bone formation following transplantation of human adipose-derived stromal cells that are not differentiated osteogenically

A number of studies have shown in vivo bone regeneration by transplantation of osteogenic cells differentiated in vitro from adipose-derived stromal cells (ADSCs). However, the in vitro osteogenic differentiation process requires an additional culture period, and the dexamethasone that is generally used in the process may be cytotoxic. Here, we tested the hypothesis that ADSCs that are not differentiated osteogenically in vitro prior to transplantation would extensively regenerate bone in vivo when exogenous bone morphogenetic protein-2 (BMP-2) is delivered to the transplantation site. We fabricated a poly(dl-lactic-co-glycolic acid)/hydroxyapatite (PLGA/HA) composite scaffold with osteoactive HA that is highly exposed on the scaffold surface. This scaffold was able to release BMP-2 over a 4-week period in vitro. Human ADSCs cultured on BMP-2-loaded PLGA/HA scaffolds for 2 weeks differentiated toward osteogenic cells expressing alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) mRNA, while cells on PLGA/HA scaffolds without BMP-2 expressed only ALP. To study in vivo bone formation, PLGA/HA scaffolds (group 1), BMP-2-loaded PLGA/HA scaffolds (group 2), undifferentiated ADSCs seeded on PLGA/HA scaffolds (group 3), and undifferentiated ADSCs seeded on BMP-2-loaded PLGA/HA scaffolds (group 4) were implanted into dorsal, subcutaneous spaces of athymic mice. Eight weeks after implantation, group 4 exhibited a 25-fold greater bone formation area and 5-fold higher calcium deposition than group 3. Bone regeneration by transplanted human ADSCs in group 4 was confirmed by expression of human-specific osteoblastic genes, ALP, collagen type I, OPN, OCN, and bone sialoprotein, while group 3 expressed much lower levels of collagen type I and OPN mRNA only. This study demonstrates the feasibility of extensive in vivo bone regeneration by transplantation of ADSCs without prior in vitro osteogenic differentiation, and that a PLGA/HA composite BMP-2 delivery system stimulates bone regeneration following transplantation of undifferentiated human ADSCs.     

RhBMP-2-loaded Poly(lactic-co-glycolic acid) microspheres fabricated by coaxial electrospraying for protein delivery

In this study, we fabricated recombinant human bone morphogenetic protein-2 (rhBMP-2) loaded Poly(lactic-co-glycolic acid) (PLGA) microspheres with core–shell structures and particle sizes ranging from 2.5 to 8 μm by coaxial electrospraying. The manufacturing process of core–shell microspheres by coaxial electrospraying is simpler than that with other methods, and a smaller diameter can be obtained. The microspheres were analyzed by environmental scanning electron microscopy, transmission electron microscopy (TEM), and laser scanning confocal microscopy (LSCM). Moreover, the drug release profiles and degradation of rhBMP-2-loaded PLGA microspheres in vitro were investigated for 21 days and for 7 weeks, respectively. The rhBMP-2 was stabilized by using bovine serum albumin (BSA) to ensure protein activity in the electrospraying process. Fluorescently labeled protein that was loaded into the core–shell PLGA microspheres was verified by LSCM. The distinct layered structure that existed in the manufactured core–shell microspheres can be observed by TEM. Cell Counting Kit-8 (CCK-8) indicated that the core–shell PLGA microspheres loaded with rhBMP-2 have great potential for the treatment of bone defects, for bone regeneration, and in bone tissue engineering.     

纤维蛋白胶复合重组人骨形态发生蛋白2微球对犬骨髓基质细胞增殖与分化的影响

背景：前期实验证实聚乳酸-聚乙醇酸微球/纤维蛋白胶能作为重组人骨形态发生蛋白2的良好可注射性缓释载体。 目的：观察可注射性骨形态发生蛋白缓释体系对犬骨髓基质细胞增殖与分化的影响。 方法：采用复乳-溶剂挥发法制备重组人骨形态发生蛋白2/聚乳酸-聚乙醇酸共聚物载药微球,然后将微球与纤维蛋白胶复合制备出重组人骨形态发生蛋白2/聚乳酸-聚乙醇酸共聚物/纤维蛋白胶复合材料,采用细胞培养及组织化学等方法观察微球对犬骨髓基质细胞的增殖与分化的影响。 结果与结论：重组人骨形态发生蛋白2/聚乳酸-聚乙醇酸共聚物/纤维蛋白胶微球对骨髓基质细胞的增殖无明显影响,但对细胞的分化功能有明显的促进作用。说明纤维蛋白胶复合重组人骨形态发生蛋白2微球能够提高骨髓基质细胞的体外成骨能力,可作为骨形态发生蛋白的良好载体。     

Effect of autologous bone marrow stromal cell seeding and bone morphogenetic protein-2 delivery on ectopic bone formation in a microsphere/poly(propylene fumarate) composite

A biodegradable microsphere/scaffold composite based on the synthetic polymer poly(propylene fumarate) (PPF) holds promise as a scaffold for cell growth and sustained delivery vehicle for growth factors for bone regeneration. The objective of the current work was to investigate the in vitro release and in vivo bone forming capacity of this microsphere/scaffold composite containing bone morphogenetic protein-2 (BMP-2) in combination with autologous bone marrow stromal cells (BMSCs) in a goat ectopic implantation model. Three composites consisting of 0, 0.08, or 8 microg BMP-2 per mg of poly(lactic-co-glycolic acid) microspheres, embedded in a porous PPF scaffold, were combined with either plasma (no cells) or culture-expanded BMSCs. PPF scaffolds impregnated with a BMP-2 solution and combined with BMSCs as well as empty PPF scaffolds were also tested. The eight different composites were implanted subcutaneously in the dorsal thoracolumbar area of goats. Incorporation of BMP-2-loaded microspheres in the PPF scaffold resulted in a more sustained in vitro release with a lower burst phase, as compared to BMP-2-impregnated scaffolds. Histological analysis after 9 weeks of implantation showed bone formation in the pores of 11/16 composites containing 8 microg/mg BMP-2-loaded microspheres with no significant difference between composites with or without BMSCs (6/8 and 5/8, respectively). Bone formation was also observed in 1/8 of the BMP-2-impregnated scaffolds. No bone formation was observed in the other conditions. Overall, this study shows the feasibility of bone induction by BMP-2 release from microspheres/scaffold composites.     

Enhancement of ectopic bone formation by bone morphogenetic protein-2 released from a heparin-conjugated poly(L-lactic-co-glycolic acid) scaffold

In this study, a heparin-conjugated poly(l-lactic-co-glycolic acid) (HP-PLGA) scaffold was developed for the sustained delivery of bone morphogenetic protein-2 (BMP-2), and then used to address the hypothesis that BMP-2 delivered from this scaffold could enhance ectopic bone formation. We found the amount of heparin conjugated to the PLGA scaffolds could be increased up to 3.2-fold by using scaffolds made from star-shaped PLGA, as compared to scaffolds made from linear PLGA, and that the release of BMP-2 from the HP-PLGA scaffold was sustained for at least 14 days in vitro. The BMP-2 released from the HP-PLGA scaffold stimulated an increase in alkaline phosphatase (ALP) activity of osteoblasts for 14 days in vitro, suggesting that the HP-PLGA scaffold delivery system releases BMP-2 in a bioactive form for a prolonged period. By contrast, BMP-2 release from unmodified (no heparin) PLGA scaffolds induced a transient increase in ALP activity for the first 3 days and a decrease thereafter. In vivo bone formation studies showed the BMP-2-loaded HP-PLGA scaffolds induced bone formation to a much greater extent than did either BMP-2-loaded unmodified PLGA scaffolds or unloaded (no BMP-2) HP-PLGA scaffolds, with 9-fold greater bone formation area and 4-fold greater calcium content in the BMP-2-loaded HP-PLGA scaffold group compared to the BMP-2-loaded unmodified PLGA scaffold group. Collectively, these results demonstrate that the HP-PLGA delivery system is capable of potentiating the osteogenic efficacy of BMP-2, and underscore its importance as a possible bone regeneration strategy.     

Physicomechanical properties of sintered scaffolds formed from porous and protein-loaded poly(DL-lactic-co-glycolic acid) microspheres for potential use in bone tissue engineering

An injectable poly(DL-lactic-co-glycolic acid) (PLGA) system comprising both porous and protein-loaded microspheres capable of forming porous scaffolds at body temperature was developed for tissue regeneration purposes. Porous and non-porous (lysozyme loaded) PLGA microspheres were formulated to represent ‘low molecular weight’ 22–34 kDa, ‘intermediate molecular weight’ (IMW) 53 kDa and ‘high molecular weight’ 84–109 kDa PLGA microspheres. The respective average size of the microspheres was directly related to the polymer molecular weight. An initial burst release of lysozyme was observed from both microspheres and scaffolds on day 1. In the case of the lysozyme-loaded microspheres, this burst release was inversely related to the polymer molecular weight. Similarly, scaffolds loaded with 1 mg lysozyme/g of scaffold exhibited an inverse release relationship with polymer molecular weight. The burst release was highest amongst IMW scaffolds loaded with 2 and 3 mg/g. Sustained lysozyme release was observed after day 1 over 50 days (microspheres) and 30 days (scaffolds). The compressive strengths of the scaffolds were found to be inversely proportional to PLGA molecular weight at each lysozyme loading. Surface analysis indicated that some of the loaded lysozyme was distributed on the surfaces of the microspheres and thus responsible for the burst release observed. Overall the data demonstrates the potential of the scaffolds for use in tissue regeneration.     

Sequential growth factor delivery from complexed microspheres for bone tissue engineering

Aim of the study was to design a 3D tissue-engineering scaffold capable of sequentially delivering two bone morphogenetic proteins (BMP). The novel delivery system consisted of microspheres of polyelectrolyte complexes of poly(4-vinyl pyridine) (P(4)VN) and alginic acid loaded with the growth factors BMP-2 and BMP-7 which themselves were loaded into the scaffolds constructed of PLGA. Microspheres carrying the growth factors were prepared using polyelectrolyte solutions with different concentrations (4-10%) to control the growth factor release rate. Release kinetics was studied using albumin as the model drug and the populations that release their contents very early and very late in the release study were selected to carry BMP-2 and BMP-7, respectively. Foam porosity changed when the microspheres were loaded. Bone marrow derived stem cells (BMSC) from rats were seeded into these foams. Alkaline phosphatase (ALP) activities were found to be lowest and cell proliferation was highest at all time points with foams carrying both the microsphere populations, regardless of BMP presence. With the present doses used neither BMP-2 nor BMP-7 delivery had any direct effect on proliferation, however, they enhanced osteogenic differentiation. Co-administration of BMP enhanced osteogenic differentiation to a higher degree than with their single administration.     

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 injectable scaffold: rhBMP-2-loaded poly(lactide-co-glycolide)/hydroxyapatite composite microspheres

Poly(lactide-co-glycolide)/hydroxyapatite(50/50) (PLGA/HA(50/50)) composite microspheres were fabricated and treated with a mixture of 0.25 M NaOH aqueous solution and ethanol (v/v = 1/1) at 37 °C. The properties of untreated and treated PLGA/HA(50/50) composite microspheres were determined and compared. The results showed that the surface roughness, HA content and hydrophilicity of the treated PLGA/HA(50/50) composite microspheres increased with treatment time. However, the treatment time should be kept within 2 h in order to maintain the shape of the PLGA/HA(50/50) microspheres. At the same time, a degradation study showed that both the untreated and treated microspheres degraded gradually with time, with the treated microspheres degrading faster in the first 4 weeks. The rhBMP-2-loaded PLGA/HA(50/50) composite microspheres were prepared by solution dipping treated PLGA/HA(50/50) composite microspheres. Mouse OCT-1 osteoblast-like cells were cultured on the untreated, treated and rhBMP-2-loaded PLGA/HA(50/50) composite microspheres and the cell affinity of the various microspheres was assessed and compared. It was found that the surface-treated PLGA/HA(50/50) composite microspheres clearly promoted osteoblast attachment, proliferation and alkaline phosphatase activity. It was considered that the hydrophilicity, osteoconductivity and surface roughness were increased by the increase in the HA component, which facilitated cell growth. Moreover, the rhBMP-2 loaded on the treated PLGA/HA(50/50) composite microspheres could be slowly released and further enhanced osteoblast differentiation. The good cell affinity and enhanced osteogenic potential of the rhBMP-2-loaded PLGA/HA composite microspheres indicate that they could be used as an injectable scaffold.     

Porous silicon oxide–PLGA composite microspheres for sustained ocular delivery of daunorubicin

A water-soluble anthracycline antibiotic drug (daunorubicin, DNR) was loaded into oxidized porous silicon (pSiO₂) microparticles and then encapsulated with a layer of polymer (poly lactide-co-glycolide, PLGA) to investigate their synergistic effects in control of DNR release. Similarly fabricated PLGA–DNR microspheres without pSiO₂, and pSiO₂ microparticles without PLGA were used as control particles. The composite microparticles synthesized by a solid-in-oil-in-water emulsion method have mean diameters of 52.33 ± 16.37 μm for PLGA–pSiO₂_21/40–DNR and the mean diameter of 49.31 ± 8.87 μm for PLGA–pSiO₂_6/20–DNR. The mean size, 26.00 ± 8 μm, of PLGA–DNR was significantly smaller, compared with the other two (P < 0.0001). Optical microscopy revealed that PLGA–pSiO₂–DNR microspheres contained multiple pSiO₂ particles. In vitro release experiments determined that control PLGA–DNR microspheres completely released DNR within 38 days and control pSiO₂–DNR microparticles (with no PLGA coating) released DNR within 14 days, while the PLGA–pSiO₂–DNR microspheres released DNR for 74 days. Temporal release profiles of DNR from PLGA–pSiO₂ composite particles indicated that both PLGA and pSiO₂ contribute to the sustained release of the payload. The PLGA–pSiO₂ composite displayed a more constant rate of DNR release than the pSiO₂ control formulation, and displayed a significantly slower release of DNR than either the PLGA or pSiO₂ formulations. We conclude that this system may be useful in managing unwanted ocular proliferation when formulated with antiproliferation compounds such as DNR.     

New scaffold for recombinant human bone morphogenetic protein-2

A bioactive and resorbable scaffold is necessary to exhibit the osteoinductive potency of recombinant human bone morphogenetic protein-2 (rhBMP-2). In a previous study, we found that synthetic octacalcium phosphate (OCP) enhances bone regeneration and is replaced by newly formed bone after it is resorbed. We hypothesized that OCP may be useful as an effective scaffold for rhBMP-2 to enhance bone regeneration. To test this hypothesis, the present study was designed to investigate whether an OCP/BMP composite implant could more effectively enhance bone regeneration. A critical-sized defect was made in a rat calvarium and 1. 15 mg of OCP combined with 10 microg of rhBMP-2 (OCP/BMP 10 microg), 2. 15 mg of OCP combined with 1 microg of rhBMP-2 (OCP/BMP 1 microg), or 3. OCP (OCP alone) was implanted into the defect and fixed at 4 or 8 weeks after implantation. The percentage of newly formed bone (n-Bone%) in the defect was determined by a histomorphometrical analysis. A statistical analysis showed that n-Bone% with OCP/BMP was significantly higher than that with OCP at both time points, whereas the difference in n-Bone% between OCP/BMP 10 microg and OCP/BMP 1 microg was not significant. The present results suggest that OCP can be used as an effective scaffold for rhBMP-2 and this OCP delivery system may be able to reduce the standard effective dose of rhBMP-2, which would be beneficial because low doses (<100 microg/g OCP) of rhBMP-2 enhance bone regeneration.