Michael Hadjiargyrou

Development of a nanostructured DNA delivery scaffold via electrospinning of PLGA and PLA–PEG block copolymers

The present work utilizes electrospinning to fabricate synthetic polymer/DNA composite scaffolds for therapeutic application in gene delivery for tissue engineering. The scaffolds are non-woven, nano-fibered, membranous structures composed predominantly of poly(lactide-co-glycolide) (PLGA) random copolymer and a poly(D,L-lactide)-poly(ethylene glycol) (PLA-PEG) block copolymer. Release of plasmid DNA from the scaffolds was sustained over a 20-day study period, with maximum release occurring at approximately 2 h. Cumulative release profiles indicated amounts released were approximately 68-80% of the initially loaded DNA. Variations in the PLGA to PLA-PEG block copolymer ratio vastly affected the overall structural morphology, as well as both the rate and efficiency of DNA release. Results indicated that DNA released directly from these electrospun scaffolds was indeed intact, capable of cellular transfection, and successfully encoded the protein beta-galactosidase. When tested under tensile loads, the electrospun polymer/DNA composite scaffolds exhibited tensile moduli of approximately 35 MPa, with approximately 45% strain initially. These values approximate those of skin and cartilage. Taken together, this work represents the first successful demonstration of plasmid DNA incorporation into a polymer scaffold using electrospinning.

Control of degradation rate and hydrophilicity in electrospun non-woven poly(D, L-lactide) nanofiber scaffolds for biomedical applications

Typical properties of poly(D,L-lactide) (PLA)-based scaffolds (films and foams), such as long degradation time, mechanical stiffness and hydrophobicity, are sometimes not suitable for biomedical applications. These properties can be substantially altered by electrospinning of PLA blends with miscible poly(lactide-co-glycolide) (PLGA) random copolymers, poly(lactide-b-ethylene glycol-b-lactide) (PLA-b-PEG-b-PLA) triblock copolymers, and a lactide (used as a hydrolytic catalyst). Electrospun scaffolds based on the multi-component PLA blends, comprised of randomly interconnected webs of sub-micron sized fibers, have a bulk density of 0.3-0.4 g/cm3. In this study, the concentration effects of PLA-b-PEG-b-PLA triblock copolymer and lactide on the cell proliferation and the hydrophilicity of electrospun scaffolds were investigated. Based on in vitro degradation study, we found that the electrospun scaffold having PLA (40 wt%), PLGA (LA/GA=50/50, 25 wt%), PLA-b-PEG-b-PLA (20 wt%), and lactide (15 wt%) underwent a rapid weight loss of approximately 65% in 7 weeks. The hydrophobicity of this membrane, as determined by contact angle measurements in a cell buffer solution, decreased by approximately 50% from 105 degrees (of an electrospun PLA scaffold) to 50 degrees. The selection of suitable chemical compositions in conjunction with the non-invasive electrospinning process is useful in the production of a new kind of biodegradable scaffolds suitable for different biomedical applications such as cell storage and delivery as well as prevention of post-surgical adhesion because of their porosity, mechanical flexibility and tunable biodegradability.

The use of low-intensity ultrasound to accelerate the healing of fractures.

Double-blind, prospective, placebo-controlled clinical trials demonstrate that healing times of fresh fractures of the radius and tibia are reduced by up to 40% with the use of low-intensity ultrasound. Animal studies indicate that low-intensity ultrasound exposure results in stronger and stiffer callus formation and in acceleration of the endochondral ossification process. Extensive clinical evidence demonstrates that ultrasound represents a safe, noninvasive method of accelerating the healing of fresh fractures of the tibia, the distal aspect of the radius, the scaphoid, and the metatarsals. Clinical studies indicate that ultrasound reduces the confounding effect of smoking and patient age on the fracture-healing process. Ultrasound requires a brief, twenty-minute, daily at-home treatment regimen and has no known contraindications. The effectiveness of low-intensity ultrasound has also been demonstrated in the clinical treatment of delayed unions and nonunions. Fracture-healing is a complex biological process that involves the spatial and temporal orchestration of numerous cell types, hundreds if not thousands of genes, and the intricate organization of an extracellular matrix, all working toward restoring the bones mechanical strength and rapid return to full function. It has often been argued that nature has optimized this process and thus it would be difficult to interventionally accelerate or augment fracture-healing. How can science conceivably improve upon 600 million years of vertebrate evolution? Nevertheless, it is just this goal that has inspired an intense effort among basic-science and clinical investigators from a vast array of biotechnology and bioengineering disciplines at academic as well as industrial laboratories, to seek a means of accelerating the healing of fractured bones. In this article, the basic-science and clinical evaluation of the use of low-intensity ultrasound is reviewed and the case is made that natures process of fracture-healing, while elegant, can be accelerated with respect to achieving the ability to support clinically relevant loads. The …

Transcriptional profiling of bone regeneration: Insight into the molecular complexity of wound repair

The healing of skeletal fractures is essentially a replay of bone development, involving the closely regulated, interdependent processes of chondrogenesis and osteogenesis. Using a rat femur model of bone healing to determine the degree of transcriptional complexity of these processes, suppressive subtractive hybridization (SSH) was performed between RNA isolated from intact bone to that of callus from post-fracture (PF) days 3, 5, 7, and 10 as a means of identifying up-regulated genes in the regenerative process. Analysis of 3,635 cDNA clones revealed 588 known genes (65.8%, 2392 clones) and 821 expressed sequence tags (ESTs) (31%, 1,127). The remaining 116 cDNAs (3.2%) yielded no homology and presumably represent novel genes. Microarrays were then constructed to confirm induction of expression and determine the temporal profile of all isolated cDNAs during fracture healing. These experiments confirmed that ∼90 and ∼80% of the subtracted known genes and ESTs are up-regulated (≥2.5-fold) during the repair process, respectively. Clustering analysis revealed subsets of genes, both known and unknown, that exhibited distinct expression patterns over 21 days (PF), indicating distinct roles in the healing process. Additionally, this transcriptional profiling of bone repair revealed a host of activated signaling molecules and even pathways (i.e. Wnt). In summary, the data demonstrate, for the fist time, that the healing process is exceedingly complex, involves thousands of activated genes, and indicates that groups of genes rather than individual molecules should be considered if the regeneration of bone is to be accelerated exogenously.

Enhancement of fracture healing by low intensity ultrasound.

Fracture healing is a highly complex regenerative process that is essentially a replay of developmental events. These events include the action of many different cell types, a myriad of proteins, and active gene expression that in the majority of cases ultimately will restore the bones natural integrity. Several biologic and biophysical approaches have been introduced to minimize delayed healing and nonunions, some with promising results. One example of such an approach is low intensity pulsed ultrasound, a noninvasive form of mechanical energy transmitted transcutaneously as high frequency acoustical pressure waves in biologic organisms. Numerous in vivo animal studies and perspective double blind placebo controlled clinical trials have shown that low intensity ultrasound is capable of accelerating and augmenting the healing of fresh fractures. Preliminary evidence suggests efficacy in the treatment of delayed healing and nonunions as well. This article reviews the animal and clinical studies that consider the effects of ultrasound on fracture healing, and the in vivo and in vitro work that strives to identify the biologic mechanism(s) responsible for the ultrasound induced enhancement of osteogenesis and fracture healing.

Temporal expression of the chondrogenic and angiogenic growth factor CYR61 during fracture repair.

The repair of a fractured bone is a complex biological event that essentially recapitulates embryonic development and requires the activity of a number of different cell types undergoing proliferation, migration, adhesion, and differentiation, while at the same time expressing a host of different genes. To identify such genes, we employed differential display and compared messenger RNA (mRNA) populations isolated from postfracture (PF) day 5 calluses to those of intact rat femurs. One such gene in which expression was up‐regulated at PF day 5 is identified as CYR61, a member of the CCN family of secreted regulatory proteins. CYR61 is a growth factor that stimulates chondrogenesis and angiogenesis. We show that its mRNA expression during fracture repair is regulated temporally, with elevated levels seen as early as PF day 3 and day 5, rising dramatically at PF day 7 and day 10, and finally declining at PF day 14 and day 21. At the highest peak of expression (PF day 7 and day 10, which correlates with chondrogenesis), CYR61 mRNA levels are approximately 10‐fold higher than those detected in intact femurs. Similarly, high protein levels are detected throughout the reparative phase of the callus, particularly in fibrous tissue and periosteum, and in proliferating chondrocytes, osteoblasts, and immature osteocytes. The secreted form of CYR61 also was detected within the newly made osteoid. No labeling was detected in hypertrophic chondrocytes or in mature cortical osteocytes. These results suggest that CYR61 plays a significant role in cartilage and bone formation and may serve as an important regulator of fracture healing.

In vitro non-viral gene delivery with nanofibrous scaffolds

Extracellular and intracellular barriers typically prevent non-viral gene vectors from having an effective transfection efficiency. Formulation of a gene delivery vehicle that can overcome the barriers is a key step for successful tissue regeneration. We have developed a novel core-shelled DNA nanoparticle by invoking solvent-induced condensation of plasmid DNA (β-galactosidase or GFP) in a solvent mixture [94% N,N-dimethylformamide (DMF) + 6% 1× TE buffer] and subsequent encapsulation of the condensed DNA globule in a triblock copolymer, polylactide-poly(ethylene glycol)-polylactide (L8E78L8), in the same solvent environment. The polylactide shell protects the encapsulated DNA from degradation during electrospinning of a mixture of encapsulated DNA nanoparticles and biodegradable PLGA (a random copolymer of lactide and glycolide) to form a nanofibrous non-woven scaffold using the same solution mixture. The bioactive plasmid DNA can then be released in an intact form from the scaffold with a controlled release rate and transfect cells in vitro.

Enhanced composite electrospun nanofiber scaffolds for use in drug delivery

The utility of nanofibrous electrospun composite scaffolds has greatly expanded over the last decade, so that they now serve as viable drug delivery vehicles for a host of different biomedical applications. The material properties of electrospun scaffolds are extremely advantageous for drug delivery, in which site-specificity and lower overall medicinal dosages lead to a potential industry-altering mechanism of delivering therapeutics. Different drugs used to predominantly treat infections and cancers can easily be incorporated and released at therapeutic dosages. Further, the inherent high porosity of these electrospun scaffolds allows for a more precisely controlled degradation which is tunable by polymer composition and fiber morphology, leading to sustained drug release. This review examines the current research and breakthrough discoveries that have elevated electrospun scaffolds to a cutting-edge technology that will dramatically alter the landscape of drug delivery.

Mechanical Modulation of Molecular Signals Which Regulate Anabolic and Catabolic Activity in Bone Tissue

Identifying the molecular mechanisms that regulate bones adaptive response to alterations in load bearing may potentiate the discovery of interventions to curb osteoporosis. Adult female mice (BALB/cByJ) were subjected to catabolic (disuse) and anabolic (45 Hz, 0.3g vibration for 10 min/day) signals, and changes in the mRNA levels of thirteen genes were compared to altered indices of bone formation. Age‐matched mice served as controls. Following 4 days of disuse, significant (P = 0.05) decreases in mRNA levels were measured for several genes, including collagen type I (−55%), osteonectin (−44%), osterix (−36%), and MMP‐2 (−36%) all of which, after 21 days, had normalized to control levels. In contrast, expression of several genes in the vibrated group, which failed to show significant changes at 4 days, demonstrated significant increases after 21 days, including inducible nitric oxide synthase (iNOS) (39%, P = 0.07), MMP‐2 (54%), and receptor activator of the nuclear factor kB ligand (RANKL) (32%). Correlations of gene expression patterns across experimental conditions and time points allowed the functional clustering of responsive genes into two distinct groups. Each clusters specific regulatory role (formation vs. resorption) was reinforced by the 60% suppression of formation rates caused by disuse, and the 55% increase in formation rates stimulated by mechanical signals (P < 0.05). These data confirm the complexity of the bone remodeling process, both in terms of the number of genes involved, their interaction and coordination of resorptive and formative activity, and the temporal sensitivity of the processes. More detailed spatial and temporal correlations between altered mRNA levels and tissue plasticity may further delineate the molecules responsible for the control of bone mass and morphology. J. Cell. Biochem. 94: 982–994, 2005.

Periostin-like-factor in osteogenesis.

Periostin‐like‐factor (PLF), an isoform related to Periostin, is expressed in bone, heart, and vascular smooth muscle cells. PLF was detected by immunostaining in mesenchymal cells in the periosteum and in osteoblasts lining trabecular bone, suggesting that PLF has a role in osteogenesis. PLF has a signal peptide and is also secreted from osteoblasts in vitro. To study the function of PLF in osteogenesis, we assessed the effect of PLF on osteoblast proliferation and differentiation in vitro and bone formation in vivo. First, to examine whether PLF regulates osteoblast proliferation in vitro, the CyQUANT cell proliferation assay was performed. PLF over‐expression by adenovirus resulted in a significantly higher rate of cell proliferation compared to controls. This finding suggests that PLF promotes osteoblast proliferation in vitro. Second, to test whether PLF mediates osteoblast differentiation in vitro, differentiation markers of osteoblasts, were assessed, including alkaline phosphatase staining and activity, von Kossa staining and calcium deposition. Over‐expression of PLF resulted in higher expression and activity of alkaline phosphatase and higher amounts of mineralization and calcium deposition compared to controls. These data suggest that PLF promotes osteoblast differentiation in vitro. Third, to investigate the role of PLF in bone formation in vivo, PLF adenovirus was injected into 6‐week‐old rat femur bone marrow. Over‐expression of PLF resulted in increased bone formation within the marrow cavity. Lastly, in a model of fracture healing, PLF expression is robustly upregulated in callus osteoblasts at post‐fracture days 7 and 14. Taken together, these findings suggests that PLF induces bone formation in vivo. We conclude that PLF stimulates bone formation in vivo possibly by promoting osteoblast proliferation and differentiation. J. Cell. Physiol. 218: 584–592, 2009.