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1.  Key changes in denervated muscles and their impact on regeneration and reinnervation 
Neural Regeneration Research  2014;9(20):1796-1809.
The neuromuscular junction becomes progressively less receptive to regenerating axons if nerve repair is delayed for a long period of time. It is difficult to ascertain the denervated muscle's residual receptivity by time alone. Other sensitive markers that closely correlate with the extent of denervation should be found. After a denervated muscle develops a fibrillation potential, muscle fiber conduction velocity, muscle fiber diameter, muscle wet weight, and maximal isometric force all decrease; remodeling increases neuromuscular junction fragmentation and plantar area, and expression of myogenesis-related genes is initially up-regulated and then down-regulated. All these changes correlate with both the time course and degree of denervation. The nature and time course of these denervation changes in muscle are reviewed from the literature to explore their roles in assessing both the degree of detrimental changes and the potential success of a nerve repair. Fibrillation potential amplitude, muscle fiber conduction velocity, muscle fiber diameter, mRNA expression levels of myogenic regulatory factors and nicotinic acetylcholine receptor could all reflect the severity and length of denervation and the receptiveness of denervated muscle to regenerating axons, which could possibly offer an important clue for surgical choices and predict the outcomes of delayed nerve repair.
PMCID: PMC4239769  PMID: 25422641
nerve regeneration; denervation; reinnervation; fibrillation potential; muscle fiber conduction velocity; muscle fiber diameter; maximal isometric force; neuromuscular junction; gene expression; neural regeneration
Polypropylene fumarate (PPF) scaffolds fabricated by rapid prototyping technique were surface modified by solution deposition of electrically conductive polypyrrole coatings with or without hydroxyapatite. Scaffolds were electrically conductive with resistivity as low as 2Ω. Scaffold characterization by Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy and thermo gravimetric analysis shows both polypyrrole and hydroxyapatite are present. Cell viability, attachment, proliferation, and differentiation were analyzed using human fetal osteoblast cells. These studies show that surface modification using hydroxyapatite improved cell attachment and proliferation of osteoblasts onto the PPF scaffolds. Alkaline phosphatase activity as a marker for osteogenic differentiation of cell to mature osteoblasts was analyzed. Our data reveal that osteoblasts maintained their phenotype on PPF scaffolds with and without coatings. Thus, these scaffolds could be appropriate candidates for our future in vivo studies.
PMCID: PMC4026939  PMID: 22051167
3.  Non-invasive monitoring of BMP-2 retention and bone formation in composites for bone tissue engineering using SPECT/CT and scintillation probes 
Non-invasive imaging can provide essential information for the optimization of new drug delivery-based bone regeneration strategies to repair damaged or impaired bone tissue. This study investigates the applicability of nuclear medicine and radiological techniques to monitor growth factor retention profiles and subsequent effects on bone formation. Recombinant human bone morphogenetic protein-2 (BMP-2, 6.5 μg/scaffold) was incorporated into a sustained release vehicle consisting of poly(lactic-co-glycolic acid) microspheres embedded in a poly(propylene fumarate) scaffold surrounded by a gelatin hydrogel and implanted subcutaneously and in 5-mm segmental femoral defects in 9 rats for a period of 56 days. To determine the pharmacokinetic profile, BMP-2 was radiolabeled with 125I and the local retention of 125I-BMP-2 was measured by single photon emission computed tomography (SPECT), scintillation probes and ex vivo scintillation analysis. Bone formation was monitored by micro-computed tomography (μCT). The scaffolds released BMP-2 in a sustained fashion over the 56-day implantation period. A good correlation between the SPECT and scintillation probe measurements was found and there were no significant differences between the non-invasive and ex-vivo counting method after 8 weeks of follow up. SPECT analysis of the total body and thyroid counts showed a limited accumulation of 125I within the body. Ectopic bone formation was induced in the scaffolds and the femur defects healed completely. In vivo μCT imaging detected the first signs of bone formation at days 14 and 28 for the orthotopic and ectopic implants, respectively, and provided a detailed profile of the bone formation rate. Overall, this study clearly demonstrates the benefit of applying non-invasive techniques in drug delivery-based bone regeneration strategies by providing detailed and reliable profiles of the growth factor retention and bone formation at different implantation sites in a limited number of animals.
PMCID: PMC3974410  PMID: 19105972
Drug delivery; Controlled release; Bone morphogenetic protein-2; Single photon emission computed; tomography; Scintillation probes; Micro-computed tomography
4.  Controlled release of vascular endothelial growth factor using poly-lactic-co-glycolic acid microspheres: In vitro characterization and application in polycaprolactone fumarate nerve conduits 
Acta biomaterialia  2011;8(2):511-518.
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 VEGF encapsulating poly-lactic-co-glycolic acid (PLGA) microspheres 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 the 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 24 hours, 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 through 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.
PMCID: PMC3972821  PMID: 22019759
microsphere; poly-lactic co-glycolic acid; vascular endothelial growth factor; bioactivity; biodegradation; nerve guide
5.  Incorporation of phosphate group modulates bone cell attachment and differentiation on oligo(polyethylene glycol) fumarate hydrogel 
Acta biomaterialia  2012;8(4):1430-1439.
In this work, we have investigated the development of a synthetic hydrogel that contains a negatively charged phosphate group for use as a substrate for bone cell attachment and differentiation in culture. The photoreactive, phosphate-containing molecule, bis(2-(methacryloyloxy)ethyl)phosphate (BP), was incorporated into oligo(polyethylene glycol) fumarate hydrogel and the mechanical, rheological and thermal properties of the resulting hydrogels were characterized. Our results showed changes in hydrogel compression and storage moduli with incorporation of BP. The modification also resulted in decreased crystallinity as recorded by differential scanning calorimetry. Our data revealed that incorporation of BP improved attachment and differentiation of human fetal osteoblast (hFOB) cells in a dose-dependent manner. A change in surface chemistry and mineralization of the phosphate-containing surfaces verified by scanning electron microscopy and energy dispersive X-ray analysis was found to be important for hFOB cell attachment and differentiation. We also demonstrated that phosphate-containing hydrogels support attachment and differentiation of primary bone marrow stromal cells. These findings suggest that BP-modified hydrogels are capable of sustaining attachment and differentiation of both bone marrow stromal cells and osteoblasts that are critical for bone regeneration.
PMCID: PMC3970912  PMID: 22277774
Hydrogel; Bone regeneration; Osteoblast; Rabbit marrow stromal cells
6.  Controlled release of doxorubicin from pH-responsive microgels 
Acta biomaterialia  2012;9(3):5438-5446.
Stimuli-responsive hydrogels have enormous potential in drug delivery applications. They can be used for site-specific drug delivery due to environmental variables in the body such as pH and temperature. In this study, we have developed pH-responsive microgels for the delivery of doxorubicin (DOX) in order to optimize its anti-tumor activity while minimizing its systemic toxicity. We used a copolymer of oligo(polyethylene glycol) fumarate (OPF) and sodium methacrylate (SMA) to fabricate the pH-responsive microgels. We demonstrated that the microgels were negatively charged, and the amounts of charge on the microgels were correlated with the SMA concentration in their formulation. The resulting microgels exhibited sensitivity to the pH and ionic strength of the surrounding environment. We demonstrated that DOX was efficiently loaded into the microgels and released in a controlled fashion via an ion-exchange mechanism. Our data revealed that the DOX release was influenced by the pH and ionic strength of the solution. Moreover, we designed a phenomenological mathematical model, based on a stretched exponential function, to quantitatively analyze the cumulative release of DOX. We found a linear correlation between the maximum release of DOX calculated from the model and the SMA concentration in the microgel formulation. The anti-tumor activity of the released DOX was assessed using a human chordoma cell line. Our data revealed that OPF–SMA microgels prolonged the cell killing effect of DOX.
PMCID: PMC3970914  PMID: 23022545
pH-responsive; Doxorubicin; Microgels; Chordoma; Oligo(polyethylene glycol) fumarate
7.  Development of Electrically Conductive Oligo(polyethylene Glycol) Fumarate-Polypyrrole Hydrogels for Nerve Regeneration 
Biomacromolecules  2010;11(11):2845-2853.
Electrically conductive hydrogel composites consisting of oligo(polyethylene glycol) fumarate (OPF) and polypyrrole (PPy) were developed for applications in nerve regeneration. OPF-PPy scaffolds were synthesized using three different anions: naphthalene-2-sulfonic acid sodium salt (NSA), dodecylbenzenesulfonic acid sodium salt (DBSA), and dioctyl sulfosuccinate sodium salt (DOSS). Scaffolds were characterized by ATR-FTIR, XPS, AFM, dynamic mechanical analysis, electrical resistivity measurements, and swelling experiments. OPF-PPy scaffolds were shown to consist of up to 25 mol% polypyrrole with a compressive modulus ranging from 265 to 323 kPa and a sheet resistance ranging from 6 to 30 × 103 Ohms/square. In vitro studies using PC12 cells showed OPF-PPy materials had no cytotoxicity and PC12 cells showed distinctly better cell attachment and an increase in the percent of neurite bearing cells on OPF-PPy materials compared to OPF. The neurite lengths of PC12 cells were significantly higher on OPF-PPyNSA and OPF-PPyDBSA. These results show that electrically conductive OPF-PPy hydrogels are promising candidates for future applications in nerve regeneration.
PMCID: PMC3947846  PMID: 20942380
hydrogel; electrical; conductive; nerve; tissue regeneration
8.  Histone deacetylase 3 is required for maintenance of bone mass during aging 
Bone  2012;52(1):296-307.
Histone deacetylase 3 (Hdac3) is a nuclear enzyme that removes acetyl groups from lysine residues in histones and other proteins to epigenetically regulate gene expression. Hdac3 interacts with bone-related transcription factors and co-factors such as Runx2 and Zfp521, and thus is poised to play a key role in the skeletal system. To understand the role of Hdac3 in osteoblasts and osteocytes, Hdac3 conditional knockout (CKO) mice were created with the Osteocalcin (OCN) promoter driving Cre expression. Hdac3 CKOOCN mice were of normal size and weight, but progressively lost trabecular and cortical bone mass with age. The Hdac3 CKOOCN mice exhibited reduced cortical bone mineralization and material properties and suffered frequent fractures. Bone resorption was lower, not higher, in the Hdac3 CKOOCN mice, suggesting that primary defects in osteoblasts caused the reduced bone mass. Indeed, reductions in bone formation were observed. Osteoblasts and osteocytes from Hdac3 CKOOCN mice showed increased DNA damage and reduced functional activity in vivo and in vitro. Thus, Hdac3 expression in osteoblasts and osteocytes is essential for bone maintenance during aging.
PMCID: PMC3513670  PMID: 23085085
Histone deacetylase; Osteocalcin-Cre; Osteoblast; Osteocyte; DNA damage
9.  A systematic review of animal models used to study nerve regeneration in tissue-engineered scaffolds 
Biomaterials  2012;33(32):8034-8039.
Research on biomaterial nerve scaffolds has been carried out for 50 years. Only three materials (collagen, polycaprolactone and polyglycollic acid) have progressed to clinical use. Pre-clinical animal models are critical for testing nerve scaffolds prior to implementation in clinical practice. We have conducted a systematic review of 416 reports in which animal models were used for evaluation of nerve regeneration into synthetic conduits. A valid animal model of nerve regeneration requires it to reproduce the specific processes that take place in regeneration after human peripheral nerve injury. No distinct animal species meets all the requirements for an ideal animal model. Certain models are well suited for understanding regenerative neurobiology while others are better for pre-clinical evaluation of efficacy. The review identified that more than 70 synthetic materials were tested in eight species using 17 different nerves. Nerve gaps ranged from 1 to 90 mm. More than 20 types of assessment methodology were used with no standardization of methods between any of the publications. The review emphasizes the urgent need for standardization or rationalization of animal models and evaluation methods for studying nerve repair.
PMCID: PMC3472515  PMID: 22889485
Peripheral nerve injury; peripheral nerve repair; nerve tube; nerve scaffold; biodegradable
10.  Osteoblast Growth and Bone Healing Response to Three Dimensional Poly(ε-caprolactone fumarate) Scaffolds 
Poly(ε-caprolactone fumarate) (PCLF) scaffold formulations were assessed as a delivery system of recombinant human bone morphogenetic protein (rhBMP-2) for bone tissue engineering. The formulations included PCLF with combinations of poly(vinyl alcohol) (PVA) and hydroxyapatite (HA). The assessments included in vitro and in vivo assays. In vitro assays validated cell attachment using a pre-osteoblast cell line (MC3T3-E1). Additionally, in vitro release profiles of rhBMP-2 from PCLF scaffolds were determined up to 21 days. Data suggested PCLF incorporated with PVA and HA accelerated rhBMP-2 release and the released protein was bioactive. For the in vivo study, a critical sized defect (CSD) model in a rabbit calvaria was used to test PCLF scaffolds. At 6 weeks post-implantation, significantly more bone formation was measured in PCLF scaffolds containing rhBMP-2 than in scaffolds without rhBMP-2. In conclusion, we demonstrated PCLF delivered biologically active rhBMP-2, promoted bone healing in a CSD and has potential as a bone tissue engineering scaffold.
PMCID: PMC3213277  PMID: 21744511
poly(ε-caprolactone fumarate); three-dimensional scaffold; rabbit calvarial critical sized defect; rhBMP-2; bone tissue engineering
11.  RNA-Dependent Protein Kinase Is Essential for 2-Methoxyestradiol-Induced Autophagy in Osteosarcoma Cells 
PLoS ONE  2013;8(3):e59406.
Osteosarcoma is the most common primary malignant bone tumor in children and young adults. Surgical resection and adjunctive chemotherapy are the only widely available options of treatment for this disease. Anti-tumor compound 2-Methoxyestradiol (2-ME) triggers cell death through the induction of apoptosis in osteosarcoma cells, but not in normal osteoblasts. In this report, we have investigated whether autophagy plays a role in 2-ME actions on osteosarcoma cells. Transmission electron microscopy imaging shows that 2-ME treatment leads to the accumulation of autophagosomes in human osteosarcoma cells. 2-ME induces the conversion of the microtubule-associated protein LC3-I to LC3-II, a biochemical marker of autophagy that is correlated with the formation of autophagosomes. Conversion to LC3-II is accompanied by protein degradation in 2-ME-treated cells. 2-ME does not induce autophagosome formation in normal primary human osteoblasts. In addition, 2-ME-dependent autophagosome formation in osteosarcoma cells requires ATG7 expression. Furthermore, 2-ME does not induce accumulation of autophagosomes in osteosarcoma cells that express dominant negative mutant RNA-dependent protein kinase (PKR) and are resistant to anti-proliferative and anti-tumor effects of 2-ME. Taken together, our study shows that 2-ME treatment induces PKR-dependent autophagy in osteosarcoma cells, and that autophagy could play an important role in 2-ME-mediated anti-tumor actions and in the control of osteosarcoma.
PMCID: PMC3602192  PMID: 23527187
12.  Reformulating Polycaprolactone Fumarate to Eliminate Toxic Diethylene Glycol: Effects of Polymeric Branching and Autoclave Sterilization on Material Properties 
Acta biomaterialia  2011;8(1):133-143.
Polycaprolactone fumarate (PCLF) is a cross-linkable derivate of polycaprolactone diol that has been shown to be an effective nerve conduit material that supports regeneration across segmental nerve defects and has warranted future clinical trials. Degradation of the previously studied PCLF (PCLFDEG) releases toxic small molecules of diethylene glycol used as the initiator for the synthesis of polycaprolactone diol. In an effort to eliminate this toxic degradation product we present a strategy for the synthesis of PCLF from either propylene glycol (PCLFPPD) or glycerol (PCLFGLY). PCLFPPD is linear and resembles the previously studied PCLFDEG, while PCLFGLY is branched and exhibits dramatically different material properties. The synthesis and characterization of their thermal, rheological, and mechanical properties are reported. The results show that the linear PCLFPPD has material properties similar to the previously studied PCLFDEG. The branched PCLFGLY exhibits dramatically lower crystalline properties resulting in lower rheological and mechanical moduli, and is therefore a more compliant material. In addition, the question of an appropriate FDA approvable sterilization method is addressed. This study shows that autoclave sterilization on PCLF materials is an acceptable sterilization method for cross-linked PCLF and has minimal effect on the PCLF thermal and mechanical properties.
PMCID: PMC3226927  PMID: 21911087
Polycaprolactone fumarate; polyester; sterilization; nerve regeneration
13.  Induction of Fracture Repair by Mesenchymal Cells Derived from Human Embryonic Stem Cells or Bone Marrow 
Development of novel therapeutic approaches to repair fracture non-unions remains a critical clinical necessity. We evaluated the capacity of human embryonic stem cell (hESC)-derived mesenchymal stem/stromal cells (MSCs) to induce healing in a fracture non-union model in rats. In addition, we placed these findings in the context of parallel studies using human bone marrow MSCs (hBM-MSCs) or a no cell control group (n = 10 to 12 per group). Preliminary studies demonstrated that both for hESC-derived MSCs and hBM-MSCs, optimal induction of fracture healing required in vitro osteogenic differentiation of these cells. Based on biomechanical testing of fractured femurs, maximum torque and stiffness were significantly greater in the hBM-MSC as compared to the control group that received no cells; values for these parameters in the hESC-derived MSC group were intermediate between the hBM-MSC and control groups, and not significantly different from the control group. However, some evidence of fracture healing was evident by X-ray in the hESC-derived MSC group. Our results thus indicate that while hESC-derived MSCs may have potential to induce fracture healing in non-unions, hBM-MSCs function more efficiently in this process. Additional studies are needed to further modify hESCs to achieve optimal fracture healing by these cells.
PMCID: PMC3179810  PMID: 21674605
embryonic stem cells; mesenchymal stem/stromal cells; osteogenic; bone repair; fracture non-union
14.  Comparison of polymer scaffolds in rat spinal cord: A step toward quantitative assessment of combinatorial approaches to spinal cord repair 
Biomaterials  2011;32(32):8077-8086.
The transected rat thoracic (T9/10) spinal cord model is a platform for quantitatively compa0ring biodegradable polymer scaffolds. Schwann cell-loaded scaffolds constructed from poly (lactic co-glycolic acid) (PLGA), poly(ε-caprolactone fumarate) (PCLF), oligo(polyethylene glycol) fumarate (OPF) hydrogel or positively charged OPF (OPF+) hydrogel were implanted into the model. We demonstrated that the mechanical properties (3-point bending and stiffness) of OPF and OPF+ hydrogels closely resembled rat spinal cord. After one month, tissues were harvested and analyzed by morphometry of neurofilament-stained sections at rostral, midlevel, and caudal scaffold. All polymers supported axonal growth. Significantly higher numbers of axons were found in PCLF (P < 0.01) and OPF+ (P < 0.05) groups, compared to that of the PLGA group. OPF+ polymers showed more centrally distributed axonal regeneration within the channels while other polymers (PLGA, PCLF and OPF) tended to show more evenly dispersed axons within the channels. The centralized distribution was associated with significantly more axons regenerating (P < 0.05). Volume of scar and cyst rostral and caudal to the implanted scaffold was measured and compared. There were significantly smaller cyst volumes in PLGA compared to PCLF groups. The model provides a quantitative basis for assessing individual and combined tissue engineering strategies.
PMCID: PMC3163757  PMID: 21803415
OPF; PLGA; PCLF; axon regeneration; spinal cord injury; Schwann cell
15.  Accuracy of Motor Axon Regeneration Across Autograft, Single Lumen, and Multichannel Poly(lactic-co-glycolic Acid) (PLGA) Nerve Tubes 
Neurosurgery  2008;63(1):144-155.
Accuracy of motor axon regeneration becomes an important issue in the development of a nerve tube for motor nerve repair. Dispersion of regeneration across the nerve tube may lead to misdirection and polyinnervation. In this study, we present a series of methods to investigate the accuracy of regeneration, which we used to compare regeneration across autografts and single lumen poly(lactic-co-glycolic acid) (PLGA) nerve tubes. We also present the concept of the multichannel nerve tube that may limit dispersion by separately guiding groups of regenerating axons.
Simultaneous tracing of the tibial and peroneal nerves with fast blue (FB) and diamidino yellow (DY), 8 weeks after repair of a 1-cm nerve gap in the rat sciatic nerve, was performed to determine the percentage of double-projecting motoneurons. Sequential tracing of the peroneal nerve with DY 1 week before and FB 8 weeks after repair was performed to determine the percentage of correctly directed peroneal motoneurons.
In the cases in which there was successful regeneration across single lumen nerve tubes, more motoneurons had double projections to both the tibial and peroneal nerve branches after single lumen nerve tube repair (21.4%) than after autograft repair (5.9%). After multichannel nerve tube repair, this percentage was slightly reduced (16.9%), although not significantly. The direction of regeneration was nonspecific after all types of repair.
Retrograde tracing techniques provide new insights into the process of regeneration across nerve tubes. The methods and data presented in this study can be used as a basis in the development of a nerve tube for motor nerve repair.
PMCID: PMC3463233  PMID: 18728579
misdirection; axon targeting; double labeling; peripheral nerve regeneration; rat sciatic nerve model; retrograde tracing
16.  COL1 C-propeptide Cleavage Site Mutations Cause High Bone Mass Osteogenesis Imperfecta 
Human mutation  2011;32(6):598-609.
Osteogenesis imperfecta (OI) is most often caused by mutations in the type I procollagen genes (COL1A1/COL1A2). We identified two children with substitutions in the type I procollagen C-propeptide cleavage site, which disrupt a unique processing step in collagen maturation and define a novel phenotype within OI. The patients have mild OI caused by mutations in COL1A1 (Patient 1: p.Asp1219Asn) or COL1A2 (Patient 2: p.Ala1119Thr), respectively. Patient 1 L1-L4 DXA z-score was +3.9 and pQCT vBMD was +3.1; Patient 2 had L1-L4 DXA z-score of 0.0 and pQCT vBMD of −1.8. Patient BMD contrasts with radiographic osteopenia and histomorphometry without osteosclerosis. Mutant procollagen processing is impaired in pericellular and in vitro assays. Patient dermal collagen fibrils have irregular borders. Incorporation of pC-collagen into matrix leads to increased bone mineralization. FT-IR imaging confirms elevated mineral/matrix ratios in both patients, along with increased collagen maturation in trabecular bone, compared to normal or OI controls. Bone mineralization density distribution revealed a marked shift toward increased mineralization density for both patients. Patient 1 has areas of higher and lower bone mineralization than controls; Patient 2’s bone matrix has a mineral content exceeding even classical OI bone. These patients define a new phenotype of high BMD OI and demonstrate that procollagen C-propeptide cleavage is crucial to normal bone mineralization.
PMCID: PMC3103631  PMID: 21344539
Osteogenesis imperfecta; C-propeptide; collagen; C-proteinase; mineralization; high bone mass
17.  Sustained Delivery of Dibutyryl Cyclic Adenosine Monophosphate to the Transected Spinal Cord Via Oligo [(Polyethylene Glycol) Fumarate] Hydrogels 
Tissue Engineering. Part A  2011;17(9-10):1287-1302.
This study describes the use of oligo [(polyethylene glycol) fumarate] (OPF) hydrogel scaffolds as vehicles for sustained delivery of dibutyryl cyclic adenosine monophosphate (dbcAMP) to the transected spinal cord. dbcAMP was encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres, which were embedded within the scaffolds architecture. Functionality of the released dbcAMP was assessed using neurite outgrowth assays in PC12 cells and by delivery to the transected spinal cord within OPF seven channel scaffolds, which had been loaded with Schwann cells or mesenchymal stem cells (MSCs). Our results showed that encapsulation of dbcAMP in microspheres lead to prolonged release and continued functionality in vitro. These microspheres were then successfully incorporated into OPF scaffolds and implanted in the transected thoracic spinal cord. Sustained delivery of dbcAMP inhibited axonal regeneration in the presence of Schwann cells but rescued MSC-induced inhibition of axonal regeneration. dbcAMP was also shown to reduce capillary formation in the presence of MSCs, which was coupled with significant functional improvements. Our findings demonstrate the feasibility of incorporating PLGA microsphere technology for spinal cord transection studies. It represents a novel sustained delivery mechanism within the transected spinal cord and provides a platform for potential delivery of other therapeutic agents.
PMCID: PMC3079174  PMID: 21198413
18.  The effects of fixed electrical charge on chondrocyte behavior 
Acta biomaterialia  2011;7(5):2080-2090.
In this study, we have compared the effects of negative and positive fixed charge on chondrocyte behavior in vitro. Electrical charges have been incorporated into oligo(poly(ethylene glycol) fumarate) (OPF) using small charged monomers such as sodium methacrylate (SMA) and (2-(methacryloyloxy) ethyl)-trimethyl ammonium chloride (MAETAC) to produce negatively and positively charged hydrogels, respectively. The hydrogel physical and electrical properties were characterized through measuring and calculating the swelling ratio and zeta potential, respectively. Our results revealed that the properties of these OPF modified hydrogels varied according to the concentration of charged monomers. Zeta potential measurements demonstrated that the electrical property of the OPF hydrogel surfaces changed due to incorporation of SMA and MAETAC and that this change in electrical property was dose-dependent. Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy was used to determine the hydrogel surface composition. To assess the effects of surface properties on chondrocyte behavior, primary chondrocytes isolated from rabbit ears were seeded as a monolayer on top of the hydrogels. We demonstrated that the cells remained viable over 7 days and began to proliferate while seeded on top of the hydrogels. Collagen type II staining was positive in all samples; however, the intensity of the stain was higher on negatively charged hydrogels. Similarly, GAG production was significantly higher on negatively charged hydrogels compared to neutral hydrogel. Reverse transcription polymerase chain reaction showed up-regulation of collagen type II and down-regulation of collagen type I on the negatively charged hydrogels. These findings indicate that charge plays an important role in establishing an appropriate environment for chondrocytes and hence in the engineering of cartilage. Thus, further investigation into charged hydrogels for cartilage tissue engineering is merited.
PMCID: PMC3103083  PMID: 21262395
hydrogel; cartilage tissue engineering; OPF; scaffold
19.  Material properties and electrical stimulation regimens through polycaprolactone fumarate-polypyrrole scaffolds as potential conductive nerve conduits 
Acta biomaterialia  2010;7(3):944-953.
Mechanical and electrical properties of polycaprolactone fumarate-polypyrrole (PCLF-PPy) scaffolds were studied under physiological conditions to evaluate their ability to maintain material properties necessary for application as conductive nerve conduits. PC12 cells cultured on PCLF-PPy scaffolds were stimulated with regimens of 10 μA of constant or 20 Hz frequency current passed through the scaffolds for 1 h/day. PC12 cellular morphologies were analyzed by fluorescence microscopy after 48 h. PCLF-PPy scaffolds exhibited excellent mechanical properties at 37°C which would allow suturing and flexibility. The surface resistivity of the scaffolds was 2kΩ and the scaffolds were electrically stable during application of electrical stimulation (ES). In vitro studies showed significant increases in percentage of neurite bearing cells, number of neurites per cell and neurite length in the presence of ES compared to no ES. Additionally, extending neurites were observed to align in the direction of the applied current. This study shows that electrically conductive PCLF-PPy scaffolds possess material properties necessary for application as nerve conduits. Additionally, the capability to significantly enhance and direct neurite extension by passing electrical current through PCLF-PPy scaffolds renders them even more promising as future therapeutic treatments for severe nerve injuries.
PMCID: PMC3031729  PMID: 20965280
Electrical Stimulation; Polypyrrole; Nerve; PCLF; PC12 cells
20.  Crosslinking Characteristics and Mechanical Properties of an Injectable Biomaterial Composed of Polypropylene fumarate and Polycaprolactone Copolymer 
In this work, a series of copolymers of polypropylene fumarate-co-polycaprolactone (PPF-co-PCL) were synthesized via a three-step polycondensation reaction of oligomeric polypropylene fumarate (PPF) with polycaprolactone (PCL). The effects of PPF precursor molecular weight, PCL precursor molecular weight, and PCL fraction in the copolymer (PCL feed ratio) on the maximum crosslinking temperature, gelation time, and mechanical properties of the crosslinked copolymers were investigated. The maximum crosslinking temperature fell between 38.2±0.3 and 47.2±0.4 °C, which increased with increasing PCL precursor molecular weight. The gelation time was between 4.2±0.2 and 8.5±0.7 min, and decreased with increasing PCL precursor molecular weight. The compressive moduli ranged from 44±1.8 to 142±7.4 MPa, with enhanced moduli at higher PPF precursor molecular weight and lower PCL feed ratio. The compressive toughness was in the range of 4.1±0.3 and 17.1±1.3 KJ/m3. Our data suggest that the crosslinking and mechanical properties of PPF-co-PCL can be modulated by varying the composition. Therefore the PPF-co-PCL copolymers may offer increased versatility as an injectable, in situ polymerizable biomaterial than the individual polymers of PPF and PCL.
PMCID: PMC3062160  PMID: 20566042
Polypropylene fumarate; polycaprolactone; injectable biomaterials; in situ polymerizable
21.  In vitro and In vivo Release of Nerve Growth Factor from Biodegradable Poly-Lactic-Co-Glycolic-Acid Microspheres 
Regeneration of peripheral nerves after injury is suboptimal. We now report the long term delivery of nerve growth factor (NGF) by biodegradable poly-lactic-co-glycolic acid (PLGA) microspheres in vitro and in vivo. Lactic to glycolic acid ratios of 50:50 and 85:15 were fabricated using the double emulsion solvent, evaporation technique. Three different inherent viscosities (0.1dL/g: 1A, 0.4dL/g: 4A, 0.7dL/g: 7A) were analyzed. In vitro, release of NGF for 23 days was measured. Electron microscopy demonstrated intact spheres for at least 7 days (50:50 1A), 14 days (50:50 4A) or 35 days (50:50 7A and 85:15 7A). In vitro release kinetics were characterized by burst release, followed by release of NGF at a rate of 0.6%-1.6% a day. Release curves for 50:50 1A and 85:15 7A differed significantly from other compositions (p<0.01). In vivo, release was characterized by a novel radionuclide tracking assay. Release rates varied from 0.9%-2.2% per day with linear kinetics. All but the 85:15 type of spheres showed different release profiles in vivo compared to in vitro conditions. Based on the surface morphology and release profiles we found microspheres fabricated from 50:50 4A PLGA to be best suited for the use in a rat sciatic nerve injury model.
PMCID: PMC2989534  PMID: 20878933
Nerve Growth Factor; Microspheres; Peripheral Nerve; Poly-lactic-co-glycolic-acid; Dorsal root ganglia
22.  Enhanced Bone Morphogenetic Protein-2-Induced Ectopic and Orthotopic Bone Formation by Intermittent Parathyroid Hormone (1–34) Administration 
Tissue Engineering. Part A  2010;16(12):3769-3777.
Bone morphogenetic proteins (BMPs) play a central role in local bone regeneration strategies, whereas the anabolic features of parathyroid hormone (PTH) are particularly appealing for the systemic treatment of generalized bone loss. The aim of the current study was to investigate whether local BMP-2-induced bone regeneration could be enhanced by systemic administration of PTH (1–34). Empty or BMP-2-loaded poly(lactic-co glycolic acid)/poly(propylene fumarate)/gelatin composites were implanted subcutaneously and in femoral defects in rats (n = 9). For the orthotopic site, empty defects were also tested. Each of the conditions was investigated in combination with daily administered subcutaneous PTH (1–34) injections in the neck. After 8 weeks of implantation, bone mineral density (BMD) and bone volume were analyzed using microcomputed tomography and histology. Ectopic bone formation and almost complete healing of the femoral defect were only seen in rats that received BMP-2-loaded composites. Additional treatment of the rats with PTH (1–34) resulted in significantly (p < 0.05) enhanced BMD and bone volume in the BMP-2 composites at both implantation sites. Despite its effect on BMD in the humerus and vertebra, PTH (1–34) treatment had no significant effect on BMD and bone volume in the empty femoral defects and the ectopically or orthotopically implanted empty composites. Histological analysis showed that the newly formed bone had a normal woven and trabecular appearance. Overall, this study suggests that intermittent administration of a low PTH dose alone has limited potential to enhance local bone regeneration in a critical-sized defect in rats. However, when combined with local BMP-2-releasing scaffolds, PTH administration significantly enhanced osteogenesis in both ectopic and orthotopic sites.
PMCID: PMC2991197  PMID: 20666615
23.  A Stimuli-Responsive Hydrogel for Doxorubicin Delivery 
Biomaterials  2010;31(31):8051-8062.
The goal of this study was to develop a polymeric carrier for delivery of anti-tumor drugs and sustained release of these agents in order to optimize anti-tumor activity while minimizing systemic effects. We used oligo(poly(ethylene glycol) fumarate) (OPF) hydrogels modified with small negatively charged molecules, sodium methacrylate (SMA), for delivery of doxorubicin (DOX). SMA at different concentrations was incorporated into the OPF hydrogel with a photo-crosslinking method. The resulting hydrogels exhibited sensitivity to the pH and ionic strength of the surrounding environment. Our results revealed that DOX was bound to the negatively charged hydrogel through electrostatic interaction and was released in a timely fashion with an ion exchange mechanism. Release kinetics of DOX was directly correlated to the concentration of SMA in the hydrogel formulations. Anti-tumor activity of the released DOX was assessed using a human osteosarcoma cell line. Our data revealed that DOX released from the modified, charged hydrogels remained biologically active and had the capability to kill cancer cells. In contrast, control groups of unmodified OPF hydrogels with or without DOX did not exhibit any cytotoxicity. This study demonstrates the feasibility of using SMA-modified OPF hydrogels as a potential carrier for chemotherapeutic drugs for cancer treatments.
PMCID: PMC2936247  PMID: 20696470
24.  Effects of composite formulation on the mechanical properties of biodegradable poly(propylene fumarate)/bone fiber scaffolds 
The objective of our study was to determine the effects of composite formulation on the compressive modulus and ultimate strength of a biodegradable, in situ polymerizable poly(propylene fumarate) (PPF) and bone fiber scaffold. The following parameters were investigated: the incorporation of bone fibers (either mineralized or demineralized), PPF molecular weight, N-vinyl pyrrolidinone (NVP) crosslinker amount, benzoyl peroxide (BP) initiator amount, and sodium chloride porogen amount. Eight formulations were chosen based on a resolution III two level fractional factorial design. The compressive modulus and ultimate strength of these formulations were measured on a materials testing machine. Absolute values for compressive modulus varied from 21.3 to 271 MPa and 2.8 to 358 MPa for dry and wet samples, respectively. The ultimate strength of the crosslinked composites varied from 2.1 to 20.3 MPa for dry samples and from 0.4 to 16.6 MPa for wet samples. Main effects of each parameter on the measured property were calculated. The incorporation of mineralized bone fibers and an increase in PPF molecular weight resulted in higher compressive modulus and ultimate strength. Both mechanical properties also increased as the amount of benzoyl peroxide increased or the NVP amount decreased in the formulation. Sodium chloride had a dominating effect on the increase of mechanical properties in dry samples but showed little effects in wet samples. Demineralization of bone fibers led to a decrease in the compressive modulus and ultimate strength. Our results suggest that bone fibers are appropriate as structural enforcement components in PPF scaffolds. The desired orthopaedic PPF scaffold might be obtained by changing a variety of composite formulation parameters.
PMCID: PMC3201805  PMID: 22034584
poly(propylene fumarate); bone fiber; orthopaedic biomaterials; injectable; mechanical properties
25.  In Vivo Biodegradation and Biocompatibility of PEG/Sebacic Acid-Based Hydrogels using a Cage Implant System 
Comprehensive in vivo biodegradability and biocompatibility of unmodified and Arg-Gly-Asp (RGD) peptide-modified PEG/Sebacic acid based hydrogels were evaluated and compared to the control material poly(lactide-co-glycolide) (PLGA) using a cage implantation system, as well as direct subcutaneous implantation for up to 12 weeks. The total weight loss after 12 weeks of implantation for unmodified PEGSDA and RGD-modified PEGSDA in the cage was approximately 42% and 52%, respectively, with no statistical difference (p> 0.05). The exudate analysis showed that PEGSDA hydrogels induced minimal inflammatory response up to 21 days following implantation, similar to the controls (empty cage and the cage containing PLGA discs). Histology analysis from direct subcutaneous implantation of the hydrogels and PLGA scaffold showed statistically similar resolution of the acute and chronic inflammatory responses with development of the fibrous capsule between the PEGSDA hydrogels and the control (PLGA). The cage system, as well as the histology analysis, demonstrated that the degradation products of both hydrogels, with or without RGD peptide modification, are biocompatible without statistically significant differences in the inflammatory responses, as compared to PLGA.
PMCID: PMC2928850  PMID: 20574982
In vivo biocompatibility; In vivo biodegradation; PEG sebacic acid diacrylate; Hydrogel; RGD-modified hydrogel; Cage implantation

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