Controlled release of bioactive TGF-beta 1 from microspheres embedded within biodegradable hydrogels

Transforming growth factor-beta1 (TGF-beta1) is of great relevance to cartilage development and regeneration. A delivery system for controlled release of growth factors such as TGF-beta1 may be therapeutic for cartilage repair. We have encapsulated TGF-beta1 into poly(DL-lactide-co-glycolide) (PLGA) microspheres, and subsequently incorporated the microspheres into biodegradable hydrogels. The hydrogels are poly(ethylene glycol) based, and the degradation rate of the hydrogels is controlled by the non-toxic cross-linking reagent, genipin. Release kinetics of TGF-beta1 were assessed using ELISA and the bioactivity of the released TGF-beta1 was evaluated using a mink lung cell growth inhibition assay. The controlled release of TGF-beta1 encapsulated within microspheres embedded in scaffolds is better controlled when compared to delivery from microspheres alone. ELISA results indicated that TGF-beta1 was released over 21 days from the delivery system, and the burst release was decreased when the microspheres were embedded in the hydrogels. The concentration of TGF-beta1 released from the gels can be controlled by both the mass of microspheres embedded in the gel, and by the concentration of genipin. Additionally, the scaffold permits containment and conformation of the spheres to the defect shape. Based on these in vitro observations, we predict that we can develop a microsphere-loaded hydrogel for controlled release of TGF-beta1 to a cartilage wound site.     

Microporous silk fibroin scaffolds embedding PLGA microparticles for controlled growth factor delivery in tissue engineering

The development of prototype scaffolds for either direct implantation or tissue engineering purposes and featuring spatiotemporal control of growth factor release is highly desirable. Silk fibroin (SF) scaffolds with interconnective pores, carrying embedded microparticles that were loaded with insulin-like growth factor I (IGF-I), were prepared by a porogen leaching protocol. Treatments with methanol or water vapor induced water insolubility of SF based on an increase in β-sheet content as analyzed by FTIR. Pore interconnectivity was demonstrated by SEM. Porosities were in the range of 70–90%, depending on the treatment applied, and were better preserved when methanol or water vapor treatments were prior to porogen leaching. IGF-I was encapsulated into two different types of poly(lactide-co-glycolide) microparticles (PLGA MP) using uncapped PLGA (50:50) with molecular weights of either 14 or 35 kDa to control IGF-I release kinetics from the SF scaffold. Embedded PLGA MP were located in the walls or intersections of the SF scaffold. Embedment of the PLGA MP into the scaffolds led to more sustained release rates as compared to the free PLGA MP, whereas the hydrolytic degradation of the two PLGA MP types was not affected. The PLGA types used had distinct effects on IGF-I release kinetics. Particularly the supernatants of the lower molecular weight PLGA formulations turned out to release bioactive IGF-I. Our studies justify future investigations of the developed constructs for tissue engineering applications.     

Biodegradable insulin-loaded PLGA microspheres fabricated by three different emulsification techniques: investigation for cartilage tissue engineering

Growth, differentiation and migration factors facilitate the engineering of tissues but need to be administered with defined gradients over a prolonged period of time. In this study insulin as a growth factor for cartilage tissue engineering and a biodegradable PLGA delivery device were used. The aim was to investigate comparatively three different microencapsulation techniques, solid-in-oil-in-water (s/o/w), water-in-oil-in-water (w/o/w) and oil-in-oil-in-water (o/o/w), for the fabrication of insulin-loaded PLGA microspheres with regard to protein loading efficiency, release and degradation kinetics, biological activity of the released protein and phagocytosis of the microspheres. Insulin-loaded PLGA microspheres prepared by all three emulsification techniques had smooth and spherical surfaces with a negative zeta potential. The preparation technique did not affect particle degradation nor induce phagocytosis by human leukocytes. The delivery of structurally intact and biologically active insulin from the microspheres was shown using circular dichroism spectroscopy and a MCF7 cell-based proliferation assay. However, the insulin loading efficiency (w/o/w about 80%, s/o/w 60%, and o/o/w 25%) and the insulin release kinetics were influenced by the microencapsulation technique. The results demonstrate that the w/o/w microspheres are most appropriate, providing a high encapsulation efficiency and low initial burst release, and thus these were finally used for cartilage tissue engineering. Insulin released from w/o/w PLGA microspheres stimulated the formation of cartilage considerably in chondrocyte high density pellet cultures, as determined by increased secretion of proteoglycans and collagen type II. Our results should encourage further studies applying protein-loaded PLGA microspheres in combination with cell transplants or cell-free in situ tissue engineering implants to regenerate cartilage.     

Sustained release of vascular endothelial growth factor from mineralized poly(lactide-co-glycolide) scaffolds for tissue engineering

Strategies to engineer bone tissue have focused on either: (1) the use of scaffolds for osteogenic cell transplantation or as conductive substrates for guided bone regeneration; or (2) release of inductive bioactive factors from these scaffold materials. This study describes an approach to add an inductive component to an osteoconductive scaffold for bone tissue engineering. We report the release of bioactive vascular endothelial growth factor (VEGF) from a mineralized, porous, degradable polymer scaffold. Three dimensional, porous scaffolds of the copolymer 85 : 15 poly(lactide-co-glycolide) were fabricated by including the growth factor into a gas foaming/particulate leaching process. The scaffold was then mineralized via incubation in a simulated body fluid. Growth of a bone-like mineral film on the inner pore surfaces of the porous scaffold is confirmed by mass increase measurements and quantification of phosphate content within scaffolds. Release of ¹²⁵I-labeled VEGF was tracked over a 15 day period to determine release kinetics from the mineralized scaffolds. Sustained release from the mineralized scaffolds was achieved, and growth of the mineral film had only a minor effect on the release kinetics from the scaffolds. The VEGF released from the mineralized and non-mineralized scaffolds was over 70% active for up to 12 days following mineralization treatment, and the growth of mineral had little effect on total scaffold porosity.     

Sustained release of ascorbate-2-phosphate and dexamethasone from porous PLGA scaffolds for bone tissue engineering using mesenchymal stem cells

The purpose of this research was to develop porous poly(D,L-lactide-co-glycolide) (PLGA) scaffolds from which ascorbate-2-phosphate (AsAP) and dexamethasone (Dex) are continuously released for a month for osteogenesis of mesenchymal stem cells for bone tissue engineering. Porous PLGA matrices containing AsAP and Dex were prepared by solvent casting/particulate leaching method. In vitro release and water uptake studies were performed in Dulbecco's phosphate buffered saline at 37 degrees C and 15 rpm. Drug loading and release rates were determined by high performance liquid chromatography. Release studies of Dex and AsAP showed that, after an initial burst release lasting 4 and 9 days, respectively, release rates followed zero order kinetics with high correlation coefficients at least until 35 days. Incorporation of AsAP into the scaffolds increased the release rates of Dex and AsAP, and the scaffold water uptake. When mesenchymal stem cells (MSCs) were cultured in the AsAP and Dex containing scaffolds in vitro, the amount of mineralization was significantly higher than in control scaffolds. In conclusion, AsAP and Dex were incorporated into porous PLGA scaffolds and continuously released over a month and osteogenesis of MSCs was increased by culture in these scaffolds.     

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.     

Modulation of protein delivery from modular polymer scaffolds

Growth factors are increasingly employed to promote tissue regeneration with various biomaterial scaffolds. In vitro release kinetics of protein growth factors from tissue engineering scaffolds are often investigated in aqueous environment, which is significantly different from in vivo environment. This study investigates the release of model proteins with net-positive (histone) and net-negative charge (bovine serum albumin, BSA) from various scaffolding surfaces and from encapsulated microspheres in the presence of ions, proteins, and cells. The release kinetics of proteins in media with varying concentrations of ions (NaCl) suggests stronger electrostatic interaction between the positively charged histone with the negatively charged substrates. While both proteins released slowly from hydrophobic PCL surfaces, plasma etching resulted in rapid release of BSA, but not histone. Interestingly, although negatively charged BSA released readily from negatively charged collagen (col), BSA released slowly from col-coated PCL scaffolds. Such electrostatic interaction effects were abolished in the presence of serum proteins and cells as evidenced by the rapid release of proteins from col-coated scaffolds. To achieve sustained release in the complex environment of serum proteins and cells, the model proteins were encapsulated into poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres, which were embedded within col-coated PCL scaffolds. Protein release from microspheres was modulated by changing the lactide-to-glycolide ratio of PLGA polymer. BSA adsorbed to col released faster than histone encapsulated in microspheres in the presence of serum and cells. Collectively, the data suggest that growth factor release is highly influenced by scaffold surface and the presence of ions, proteins, and cells in the media. Strategies to deliver multiple growth factors and studies which investigate their release should consider these important variables.     

Retention and activity of BMP-2 in hyaluronic acid-based scaffolds in vitro.

Bone morphogenetic protein-2 (BMP-2) delivered in a suitable implantable matrix has the potential to repair local skeletal defects by inducing new bone formation from undifferentiated pluripotent stem cells resident in host tissue. In this study, we examined in vitro the potential of a derivatized hyaluronic acid (Hyaff-11) scaffold as a delivery vehicle for recombinant human BMP-2 (rhBMP-2) in bone and cartilage repair therapies. Hyaff-11 scaffolds were fabricated using a phase inversion/particulate leaching method and soak-loaded with rhBMP-2. In vitro release kinetics of rhBMP-2, demonstrated using enzyme-linked immunosorbant assay and alkaline phosphatase (ALP) assay revealed a slow, sustained rhBMP-2 release during 28 days, with a cumulative release of 31.82% of the initial rhBMP-2 loaded. rhBMP-2 was released in bioactive form as demonstrated by ALP induction of pluripotent cell line, C3H10T1/2 (T1/2), down the osteoblast lineage when incubated with the release supernatants. rhBMP-2 retention in Hyaff-11 scaffolds was greater than that from collagen gels, which released most of the initially loaded rhBMP-2 by 14 days. rhBMP-2-loaded Hyaff-11 scaffolds were also seeded with T1/2 cells and evaluated at 3, 7, 14, and 28 days for viability and expression of osteoblast phenotype. Cells remained viable throughout the study and expressed a time- and dose-dependent ALP and osteocalcin expression in the rhBMP-2 groups. Based on these observations, Hyaff-11 scaffolds may be suitable delivery systems for rhBMP-2 in bone/cartilage repair because of their ability to retain rhBMP-2, release low levels of bioactive rhBMP-2 to the local environment in a sustained manner, and stimulate differentiation of pluripotent stem cells.     

Controlled release of transforming growth factor beta1 from biodegradable polymer microparticles

Recombinant human transforming growth factor beta1 (TGF-beta1) was incorporated into biodegradable microparticles of blends of poly(DL-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) at 6 ng/1 mg microparticles. Fluorescein isothiocynate labeled bovine serum albumin (FITC-BSA) was coencapsulated as a porogen at 4 microg/1 mg of microparticles. The effects of PEG content (0, 1, or 5 wt %) and buffer pH (3, 5, or 7.4) on the protein release kinetics and the degradation of PLGA were determined in vitro for up to 28 days. The entrapment yield of TGF-beta1 was 83.4 +/- 13.1 and 54.2 +/- 12.1% for PEG contents of 0 and 5%, respectively. The FITC-BSA and TGF-beta1 were both released in a multiphasic fashion including an initial burst effect. Increasing the PEG content resulted in the decreased cumulative mass of released proteins. By day 28, 3.8 +/- 0. 1 and 2.8 +/- 0.3 microg (based on 1 mg microparticles) of loaded FITC-BSA and 3.4 +/- 0.2 and 2.2 +/- 0.3 ng of loaded TGF-beta1 were released into pH 7.4 phosphate buffered saline (PBS) from microparticles with 0 and 5% PEG, respectively. Aggregation of FITC-BSA occurred at lower buffer pH, which led to decreased release rates of both proteins. For microparticles with 5% PEG, 2.3 +/- 0.1 microg of FITC-BSA and 2.0 +/- 0.2 ng of TGF-beta1 were released in pH 7.4 buffer after 28 days, while only 1.7 +/- 0.3 microg and 1.3 +/- 0.4 ng of the corresponding proteins were released in pH 3 buffer. The degradation of PLGA was also enhanced at 5% PEG content, which was significantly accelerated at acidic pH conditions. The calculated half-lives of PLGA were 20.3 +/- 0.9 and 15.9 +/- 1.2 days for PEG contents of 0 and 5%, respectively, in pH 7.4 PBS and 14.8 +/- 0.4 and 5.5 +/- 0.1 days for 5% PEG in pH 7.4 and 3 buffers, respectively. These results suggest that PLGA/PEG blend microparticles are useful as delivery vehicles for controlled release of growth factors.     

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.     

Improved small molecule drug release from in situ forming poly(lactic-co-glycolic acid) scaffolds incorporating poly(β-amino ester) and hydroxyapatite microparticles

In situ forming implants are an attractive choice for controlled drug release into a fixed location. Currently, rapidly solidifying solvent exchange systems suffer from a high initial burst, and sustained release behavior is tied to polymer precipitation and degradation rate. The present studies investigated addition of hydroxyapatite (HA) and drug-loaded poly(β-amino ester) (PBAE) microparticles to in situ forming poly(lactic-co-glycolic acid) (PLGA)-based systems to prolong release and reduce burst. PBAEs were synthesized, imbibed with simvastatin (osteogenic) or clodronate (anti-resorptive), and then ground into microparticles. Microparticles were mixed with or without HA into a PLGA solution, and the mixture was injected into buffer, leading to precipitation and creating solid scaffolds with embedded HA and PBAE microparticles. Simvastatin release was prolonged through 30?days, and burst release was reduced from 81 to 39% when loaded into PBAE microparticles. Clodronate burst was reduced from 49 to 32% after addition of HA filler, but release kinetics were unaffected after loading into PBAE microparticles. Scaffold dry mass remained unchanged through day 15, with a pronounced increase in degradation rate after day 30, while wet scaffolds experienced a mass increase through day 25 due to swelling. Porosity and pore size changed throughout degradation, likely due to a combination of swelling and degradation. The system offers improved release kinetics, multiple release profiles, and rapid solidification compared to traditional in situ forming implants.     

Transforming growth factor-beta1 release from a porous electrostatic spray deposition-derived calcium phosphate coating

This study evaluated the utilization of a porous coating, derived with electrostatic spray deposition (ESD), as a carrier material for transforming growth factor-beta1 (TGF-beta1). A porous beta-tricalcium phosphate coating was deposited with ESD, and 10 ng of (125) I-labeled TGF-beta1 was loaded on the substrates. A burst release during the first hour of incubation of >90% was observed, in either culture medium or phosphate-buffered saline (PBS). Ninety-nine percent of the growth factor was released after 10 days of incubation. All samples were able to inhibit epithelial cell growth, indicating that the growth factor had remained bioactive after release. Thereafter, osteoblast-like cells were seeded upon substrates with or without 10 ng of TGF-beta1. While proliferation of osteoblast-like cells was increased on TGF-beta1-loaded substrates, differentiation was inhibited or delayed. In conclusion, a porous ESD-derived calcium phosphate coating can be used as a carrier material for TGF-beta1, when a burst release is desired.     

Dexamethasone-releasing biodegradable polymer scaffolds fabricated by a gas-foaming/salt-leaching method

Dexamethasone, a steroidal anti-inflammatory drug, was incorporated into porous biodegradable polymer scaffolds for sustained release. The slowly released dexamethasone from the degrading scaffolds was hypothesized to locally modulate the proliferation and differentiation of various cells. Dexamethasone containing porous poly(D,L-lactic-co-glycolic acid) (PLGA) scaffolds were fabricated by a gas-foaming/salt-leaching method. Dexamethasone was loaded within the polymer phase of the PLGA scaffold in a molecularly dissolved state. The loading efficiency of dexamethasone varied from 57% to 65% depending on the initial loading amount. Dexamethasone was slowly released out in a controlled manner for over 30 days without showing an initial burst release. Release amount and duration could be adjusted by controlling the initial loading amount within the scaffolds. Released dexamethasone from the scaffolds drastically suppressed the proliferations of lymphocytes and smooth muscle cells in vitro. This study suggests that dexamethasone-releasing PLGA scaffolds could be potentially used either as an anti-inflammatory porous prosthetic device or as a temporal biodegradable stent for reducing intimal hyperplasia in restenosis.     

Evaluation of chondrocyte growth in the highly porous scaffolds made by fused deposition manufacturing (FDM) filled with type II collagen

Highly porous poly(d,l-lactide-co-glycolide) (PLGA) scaffolds for cartilage tissue engineering were fabricated in this study using the fused deposition manufacturing (FDM) process and were further modified by type II collagen. The average molecular weight of PLGA decreased to about 60% of the original value after the melt-extrusion process. Type II collagen exhibited sponge-like structure and filled the macroporous FDM scaffolds. An increase of the fiber spacing resulted in an increase of the porosity. The storage modulus of FDM scaffolds with a large fiber spacing was comparable to that of the native porcine articular cartilage. Although the FDM hybrid scaffolds were swollen in various extents after 28 days of in vitro culture, the seeded chondrocytes were well distributed in the interior of the scaffolds with a large fiber spacing and neocartilage was formed around the scaffolds. The study also suggested that a low processing temperature may be required to produce PLGA precision scaffolds using FDM.     

Effects of the controlled-released TGF-beta 1 from chitosan microspheres on chondrocytes cultured in a collagen/chitosan/glycosaminoglycan scaffold

The objectives of this study were (1) to develop a three-dimensional collagen/chitosan/glycosaminoglycan (GAG) scaffold in combination with transforming growth factor-beta1 (TGF-beta 1)-loaded chitosan microspheres, and (2) to evaluate the effect of released TGF-beta 1 on the chondrogenic potential of rabbit chondrocytes in such scaffolds. TGF-beta 1 was loaded into chitosan microspheres using an emulsion-crosslinking method. The controlled release of TGF-beta 1, as measured by enzyme-linked immunosorbent assay (ELISA), was monitored for 7 days. The porous scaffolds containing collagen and chitosan were fabricated by using a freeze drying technique and crosslinked using 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC) in the presence of chondroitin sulfate (CS), as a GAG component. The TGF-beta 1 microspheres were encapsulated into the scaffold at a concentration of 10 ng TGF-beta 1/scaffold and then chondrocytes were seeded in the scaffold and incubated in vitro for 3 weeks. Both proliferation rate and glycosaminoglycan (GAG) production were significantly higher in the TGF-beta 1 microsphere-incorporated scaffolds than in the control scaffolds without microspheres. Extracellular matrix staining by Safranin O and immunohistochemistry for type II collagen were elevated in the scaffold with TGF-beta 1 microspheres. These results suggest that TGF-beta 1 microspheres when incorporated into a scaffold have the potential to enhance cartilage formation.     

Controlled release of plasmid DNA from biodegradable scaffolds fabricated using a thermally-induced phase-separation method

Highly porous poly(D,L-lactic-co-glycolic acid) (PLGA) scaffolds were fabricated by a thermally-induced phase-separation (TIPS) method to deliver plasmid DNA in a controlled manner. A variety of TIPS parameters directly affecting pore structures and their interconnectivities of the scaffold, such as polymer concentration, solvent/non-solvent ratio, quenching methods and annealing time, were systematically examined to explore their effects on sustained release behaviors of plasmid DNA. Plasmid DNA was directly loaded into the inner pore region of the scaffold during the TIPS process. By optimizing the parameters, PLGA scaffolds releasing plasmid DNA over 21 days were successfully fabricated. DNA release profiles were mainly affected by the pore structures and their interconnectivities of the scaffolds. Plasmid DNA released from the scaffolds fully maintained its structural integrity and showed comparable transfection efficiency to native plasmid DNA. These biodegradable polymeric scaffolds capable of sustained DNA release can be potentially applied for various tissue engineering purposes requiring a combined gene delivery strategy.     

Drug-releasing scaffolds fabricated from drug-loaded microspheres.

Biodegradable scaffolds serve a central role in many strategies for engineering tissue replacements or in guiding tissue regeneration. Typically, these scaffolds function to create and maintain a space and to provide a support for cell adhesion. However, these scaffolds also can serve as vehicles for the delivery of bioactive factors (e.g., protein or DNA) in order to manipulate cellular processes within the scaffold microenvironment. This study presents a novel approach to fabricate tissue-engineering scaffolds capable of sustained drug delivery whereby drug-loaded microspheres are fabricated into structures with controlled porosity. A double-emulsion process was used to fabricate microspheres with encapsulated DNA that retained its integrity and was released from the microspheres within 24 h. These DNA-loaded microspheres subsequently were formed into a nonporous disk or an interconnected open-pore scaffold (>94% porosity) via a gas-foaming process. The disks and scaffolds exhibited sustained plasmid release for at least 21 days and had minimal burst during the initial phase of release. This approach of assembling drug-loaded microspheres into porous and nonporous structures may find great utility in the fabrication of synthetic matrices that direct tissue formation.     

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.     

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.     

Fabrication of precision scaffolds using liquid-frozen deposition manufacturing for cartilage tissue engineering

The fused deposition manufacturing (FDM) system has been used to fabricate tissue-engineered scaffolds with highly interconnecting and controllable pore structure, although the system is limited to a few materials. For this reason, the liquid-frozen deposition manufacturing (LFDM) system based on an improvement of the FDM process was developed. Poly(D,L-lactide-co-glycolide) (PLGA) precision scaffolds were fabricated using LFDM from PLGA solutions of different concentrations. A greater concentration of PLGA solution resulted in greater mechanical strength but also resulted in less water content and smaller pore size on the surface of the scaffolds. LFDM scaffolds in general had mechanical strength closer to that of native articular cartilage than did FDM scaffolds. Neocartilage formation was observed in LFDM scaffolds seeded with porcine articular chondrocytes after 28 days of culture. Chondrocytes in LFDM scaffolds made from low concentrations (15-20%) of PLGA solution maintained a round shape, proliferated well, and secreted abundant extracellular matrix. In contrast, the FDM PLGA scaffolds had low cell numbers and poor matrix production because of heavy swelling. The LFDM system offered a useful way to fabricate scaffolds for cartilage tissue-engineering applications.