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author:("kraszewski, Michael")
1.  Ligament Tissue Engineering Using a Novel Porous Polycaprolactone Fumarate Scaffold and Adipose Tissue-Derived Mesenchymal Stem Cells Grown in Platelet Lysate 
Tissue Engineering. Part A  2015;21(21-22):2703-2713.
Purpose: Surgical reconstruction of intra-articular ligament injuries is hampered by the poor regenerative potential of the tissue. We hypothesized that a novel composite polymer “neoligament” seeded with progenitor cells and growth factors would be effective in regenerating native ligamentous tissue.
Methods: We synthesized a fumarate-derivative of polycaprolactone fumarate (PCLF) to create macro-porous scaffolds to allow cell–cell communication and nutrient flow. Clinical grade human adipose tissue-derived human mesenchymal stem cells (AMSCs) were cultured in 5% human platelet lysate (PL) and seeded on scaffolds using a dynamic bioreactor. Cell growth, viability, and differentiation were examined using metabolic assays and immunostaining for ligament-related markers (e.g., glycosaminoglycans [GAGs], alkaline phosphatase [ALP], collagens, and tenascin-C).
Results: AMSCs seeded on three-dimensional (3D) PCLF scaffolds remain viable for at least 2 weeks with proliferating cells filling the pores. AMSC proliferation rates increased in PL compared to fetal bovine serum (FBS) (p < 0.05). Cells had a low baseline expression of ALP and GAG, but increased expression of total collagen when induced by the ligament and tenogenic growth factor fibroblast growth factor 2 (FGF-2), especially when cultured in the presence of PL (p < 0.01) instead of FBS (p < 0.05). FGF-2 and PL also significantly increased immunostaining of tenascin-C and collagen at 2 and 4 weeks compared with human fibroblasts.
Summary: Our results demonstrate that AMSCs proliferate and eventually produce a collagen-rich extracellular matrix on porous PCLF scaffolds. This novel scaffold has potential in stem cell engineering and ligament regeneration.
doi:10.1089/ten.tea.2015.0183
PMCID: PMC4652223  PMID: 26413793
2.  Hydrolysable core crosslinked particle for receptor-mediated pH-sensitive anticancer drug delivery 
Biodegradable micelle systems with both extracellular stabilities and specific targeting properties are highly desirable for anti-cancer drug delivery. Here, we report a biodegradable and crosslinkable poly(propylene fumarate)-co-poly(lactide-co-glycolide)-co-poly(ethylene glycol) (PPF-PLGA-PEG) copolymer conjugated with folate (FA) molecules for receptor-mediated delivery of doxorubicin. Micelles with folate ligands on surface and fumarate bonds within the core were self-assembled and crosslinked, which exhibited better stability against potential physiological conditions during and after drug administration. A pH sensitive drug release profile was observed showing robust release at acidic environment due to the ester hydrolysis of PLGA (50:50). Further, micelles with folate ligands on surface showed strong targeting ability and therapeutic efficacy through receptor-mediated endocytosis, as evidenced by efficacious cancer killing and fatal DNA damage. These results imply promising potential for ligand-conjugated core crosslinked PPF-PLGA-PEG-FA micelles as carrier system for targeted anti-cancer drug delivery.
doi:10.1039/C5NJ01404B
PMCID: PMC4846283  PMID: 27134519
3.  Efficacy of Electrical Stimulators for Bone Healing: A Meta-Analysis of Randomized Sham-Controlled Trials 
Scientific Reports  2016;6:31724.
Electrical stimulation is a common adjunct used to promote bone healing; its efficacy, however, remains uncertain. We conducted a meta-analysis of randomized sham-controlled trials to establish the efficacy of electrical stimulation for bone healing. We identified all trials randomizing patients to electrical or sham stimulation for bone healing. Outcomes were pain relief, functional improvement, and radiographic nonunion. Two reviewers assessed eligibility and risk of bias, performed data extraction, and rated the quality of the evidence. Fifteen trials met our inclusion criteria. Moderate quality evidence from 4 trials found that stimulation produced a significant improvement in pain (mean difference (MD) on 100-millimeter visual analogue scale = −7.7 mm; 95% CI −13.92 to −1.43; p = 0.02). Two trials found no difference in functional outcome (MD = −0.88; 95% CI −6.63 to 4.87; p = 0.76). Moderate quality evidence from 15 trials found that stimulation reduced radiographic nonunion rates by 35% (95% CI 19% to 47%; number needed to treat = 7; p < 0.01). Patients treated with electrical stimulation as an adjunct for bone healing have less pain and are at reduced risk for radiographic nonunion; functional outcome data are limited and requires increased focus in future trials.
doi:10.1038/srep31724
PMCID: PMC4990885  PMID: 27539550
4.  Positively Charged Oligo[Poly(Ethylene Glycol) Fumarate] Scaffold Implantation Results in a Permissive Lesion Environment after Spinal Cord Injury in Rat 
Tissue Engineering. Part A  2015;21(13-14):2099-2114.
Positively charged oligo[poly(ethylene glycol) fumarate] (OPF+) scaffolds loaded with Schwann cells bridge spinal cord injury (SCI) lesions and support axonal regeneration in rat. The regeneration achieved is not sufficient for inducing functional recovery. Attempts to increase regeneration would benefit from understanding the effects of the scaffold and transplanted cells on lesion environment. We conducted morphometric and stereological analysis of lesions in rats implanted with OPF+ scaffolds with or without loaded Schwann cells 1, 2, 3, 4, and 8 weeks after thoracic spinal cord transection. No differences were found in collagen scarring, cyst formation, astrocyte reactivity, myelin debris, or chondroitin sulfate proteoglycan (CSPG) accumulation. However, when scaffold-implanted animals were compared with animals with transection injuries only, these barriers to regeneration were significantly reduced, accompanied by increased activated macrophages/microglia. This distinctive and regeneration permissive tissue reaction to scaffold implantation was independent of Schwann cell transplantation. Although the tissue reaction was beneficial in the short term, we observed a chronic fibrotic host response, resulting in scaffolds surrounded by collagen at 8 weeks. This study demonstrates that an appropriate biomaterial scaffold improves the environment for regeneration. Future targeting of the host fibrotic response may allow increased axonal regeneration and functional recovery.
doi:10.1089/ten.tea.2015.0019
PMCID: PMC4507127  PMID: 25891264
5.  ASTM International Workshop on Standards & Measurements for Tissue Engineering Scaffolds 
The “Workshop on Standards & Measurements for Tissue Engineering Scaffolds” was held on May 21, 2013 in Indianapolis, IN and was sponsored by the ASTM International (ASTM). The purpose of the workshop was to identify the highest priority items for future standards work for scaffolds used in the development and manufacture of tissue engineered medical products (TEMPs). Eighteen speakers and 78 attendees met to assess current scaffold standards and to prioritize needs for future standards. A key finding was that the ASTM TEMPs subcommittees (F04.41-46) have many active “guide” documents for educational purposes, but that few standard “test methods” or “practices” have been published. Overwhelmingly, the most clearly identified need was standards for measuring the structure of scaffolds, followed by standards for biological characterization, including in vitro testing, animal models and cell-material interactions. The third most pressing need was to develop standards for assessing the mechanical properties of scaffolds. Additional needs included standards for assessing scaffold degradation, clinical outcomes with scaffolds, effects of sterilization on scaffolds, scaffold composition and drug release from scaffolds. Discussions also highlighted the need for additional scaffold reference materials and the need to use them for measurement traceability. Finally, dialogue emphasized the needs to promote the use of standards in scaffold fabrication, characterization, and commercialization and to assess the use and impact of standards in the TEMPs community. Many scaffold standard needs have been identified and focus should now turn to generating these standards to support the use of scaffolds in TEMPs.
doi:10.1002/jbm.b.33286
PMCID: PMC4886474  PMID: 25220952
ASTM International; documentary standards; reference materials; tissue scaffold; workshop report
6.  Treatment Planning and Fracture Prediction in Patients with Skeletal Metastasis with CT-based Rigidity Analysis 
Background
Pathological fractures could be prevented if reliable methods of fracture risk assessment were available. A multi-center, prospective study was conducted to identify significant predictors of physicians' treatment plan for skeletal metastasis based on clinical fracture risk assessments and the proposed CT-based Rigidity Analysis (CTRA).
Methods
Orthopaedic oncologists selected a treatment plan for 124 patients with 149 metastatic lesions based on Mirels method. Then, CTRA was performed and the results were provided to the physicians, who were asked to reassess their treatment plan. The pre- and post-CTRA treatment plans were compared to identify cases where the treatment plan was changed based on the CTRA report. Patients were followed for a 4 month period to establish the incidence of pathological fractures.
Results
Pain, lesion type and lesion size were significant predictors of the pre-CTRA plan. After providing the CTRA results, physicians changed their plan for 36 patients. CTRA results, pain and primary source of metastasis were significant predictors of the post-CTRA plan. Follow up of patients who did not undergo fixation resulted in 7 fractures; CTRA predicted these fractures with 100% sensitivity and 90% specificity, whereas the Mirels method was 71% sensitive and 50% specific.
Conclusions
Lesion type and size and pain level influenced the physicians’ plans for management of metastatic lesions. Physicians’ treatment plans and fracture risk predictions were significantly influenced by the availability of CTRA results. Due to its high sensitivity and specificity. CTRA could potentially be used as a screening method for pathological fractures.
doi:10.1158/1078-0432.CCR-14-2668
PMCID: PMC4452435  PMID: 25724521
7.  Tunable tissue scaffolds fabricated by in situ crosslink in phase separation system 
RSC advances  2015;5(122):100824-100833.
Three-dimensional (3-D) scaffolds with intrinsic porous structures are desirable in various tissue regeneration applications. In this study, a unique method that combines thermally induced phase separation with a photocrosslinking process was developed for the fabrication of 3-D crosslinked polymer scaffolds with densely interconnected porous structures. Biodegradable poly(propylene fumarate)-co-poly(L-lactic acid) with crosslinkable fumarate bonds were used as the structural polymer material and a dioxane/water binary system was applied for the phase separation. By altering the polymer composition (9, 5 and 3 wt%), different types of scaffolds with distinct morphology, mechanical strength, degradation rate, cell growth and morphology, and extracellular matrix production were fabricated. These crosslinked 3-D porous scaffolds with tunable strength and biological responses show promise for potential applications in regenerative therapies, including bone and neural tissue engineering.
doi:10.1039/C5RA19406G
PMCID: PMC4792294  PMID: 26989479
8.  Novel biodegradable poly(propylene fumarate)-co-poly(l-lactic acid) porous scaffolds fabricated by phase separation for tissue engineering applications 
RSC advances  2015;5(27):21301-21309.
Scaffolds with intrinsically interconnected porous structures are highly desirable in tissue engineering and regenerative medicine. In this study, three-dimensional polymer scaffolds with highly interconnected porous structures were fabricated by thermally induced phase separation of novel synthesized biodegradable poly(propylene fumarate)-co-poly(l-lactic acid) in a dioxane/water binary system. Defined porous scaffolds were achieved by optimizing conditions to attain interconnected porous structures. The effect of phase separation parameters on scaffold morphology were investigated, including polymer concentration (1, 3, 5, 7, and 9%), quench time (1, 4, and 8 min), dioxane/water ratio (83/17, 85/15, and 87/13 wt/wt), and freeze temperature (−20, −80, and −196 °C). Interesting pore morphologies were created by adjusting these processing parameters, e.g., flower-shaped (5%; 85/15; 1 min; −80 °C), spherulite-like (5%; 85/15; 8 min; −80 °C), and bead-like (5%; 87/13; 1 min; −80 °C) morphology. Modulation of phase separation conditions also resulted in remarkable differences in scaffold porosities (81% to 91%) and thermal properties. Furthermore, scaffolds with varied mechanic strengths, degradation rates, and protein adsorption capabilities could be fabricated using the phase separation method. In summary, this work provides an effective route to generate multi-dimensional porous scaffolds that can be applied to a variety of hydrophobic polymers and copolymers. The generated scaffolds could potentially be useful for various tissue engineering applications including bone tissue engineering.
doi:10.1039/C5RA00508F
PMCID: PMC4792309  PMID: 26989483
9.  Tissue Engineered Constructs: Perspectives on Clinical Translation 
Annals of biomedical engineering  2015;43(3):796-804.
In this article, a “bedside to bench and back” approach for developing tissue engineered medical products (TEMPs) for clinical applications is reviewed. The driving force behind this approach is unmet clinical needs. Preclinical research, both in vitro and in vivo using small and large animal models, will help find solutions to key research questions. In clinical research, ethical issues regarding the use of cells and tissues, their sources, donor consent, as well as clinical trials are important considerations. Regulatory issues, at both institutional and government levels, must be addressed prior to the translation of TEMPs to clinical practice. TEMPs are regulated as drugs, biologics, devices, or combination products by the US Food and Drug Administration (FDA). Depending on the mode of regulation, applications for TEMP introduction must be filed with the FDA to demonstrate safety and effectiveness in premarket clinical studies, followed by 510(k) premarket clearance or premarket approval (for medical devices), biologics license application approval (for biologics), or New Drug Application approval (for drugs). A case study on nerve cuffs is presented to illustrate the regulatory process. Finally, perspectives on commercialization such as finding a company partner and funding issues, as well as physician culture change, are presented.
doi:10.1007/s10439-015-1280-0
PMCID: PMC4394896  PMID: 25711151
Tissue engineered medical products (TEMPs); bioethics; regulatory issues; Food and Drug Administration (FDA); medical devices; commercialization
10.  Controlled Delivery of Vancomycin via Charged Hydrogels 
PLoS ONE  2016;11(1):e0146401.
Surgical site infection (SSI) remains a significant risk for any clean orthopedic surgical procedure. Complications resulting from an SSI often require a second surgery and lengthen patient recovery time. The efficacy of antimicrobial agents delivered to combat SSI is diminished by systemic toxicity, bacterial resistance, and patient compliance to dosing schedules. We submit that development of localized, controlled release formulations for antimicrobial compounds would improve the effectiveness of prophylactic surgical wound antibiotic treatment while decreasing systemic side effects. Our research group developed and characterized oligo(poly(ethylene glycol)fumarate) / sodium methacrylate (OPF/SMA) charged copolymers as biocompatible hydrogel matrices. Here, we report the engineering of this copolymer for use as an antibiotic delivery vehicle in surgical applications. We demonstrate that these hydrogels can be efficiently loaded with vancomycin (over 500 μg drug per mg hydrogel) and this loading mechanism is both time- and charge-dependent. Vancomycin release kinetics are shown to be dependent on copolymer negative charge. In the first 6 hours, we achieved as low as 33.7% release. In the first 24 hours, under 80% of total loaded drug was released. Further, vancomycin release from this system can be extended past four days. Finally, we show that the antimicrobial activity of released vancomycin is equivalent to stock vancomycin in inhibiting the growth of colonies of a clinically derived strain of methicillin-resistant Staphylococcus aureus. In summary, our work demonstrates that OPF/SMA hydrogels are appropriate candidates to deliver local antibiotic therapy for prophylaxis of surgical site infection.
doi:10.1371/journal.pone.0146401
PMCID: PMC4711919  PMID: 26760034
11.  Comparison of Cellular Architecture, Axonal Growth, and Blood Vessel Formation Through Cell-Loaded Polymer Scaffolds in the Transected Rat Spinal Cord 
Tissue Engineering. Part A  2014;20(21-22):2985-2997.
The use of multichannel polymer scaffolds in a complete spinal cord transection injury serves as a deconstructed model that allows for control of individual variables and direct observation of their effects on regeneration. In this study, scaffolds fabricated from positively charged oligo[poly(ethylene glycol)fumarate] (OPF+) hydrogel were implanted into rat spinal cords following T9 complete transection. OPF+ scaffold channels were loaded with either syngeneic Schwann cells or mesenchymal stem cells derived from enhanced green fluorescent protein transgenic rats (eGFP-MSCs). Control scaffolds contained extracellular matrix only. The capacity of each scaffold type to influence the architecture of regenerated tissue after 4 weeks was examined by detailed immunohistochemistry and stereology. Astrocytosis was observed in a circumferential peripheral channel compartment. A structurally separate channel core contained scattered astrocytes, eGFP-MSCs, blood vessels, and regenerating axons. Cells double-staining with glial fibrillary acid protein (GFAP) and S-100 antibodies populated each scaffold type, demonstrating migration of an immature cell phenotype into the scaffold from the animal. eGFP-MSCs were distributed in close association with blood vessels. Axon regeneration was augmented by Schwann cell implantation, while eGFP-MSCs did not support axon growth. Methods of unbiased stereology provided physiologic estimates of blood vessel volume, length and surface area, mean vessel diameter, and cross-sectional area in each scaffold type. Schwann cell scaffolds had high numbers of small, densely packed vessels within the channels. eGFP-MSC scaffolds contained fewer, larger vessels. There was a positive linear correlation between axon counts and vessel length density, surface density, and volume fraction. Increased axon number also correlated with decreasing vessel diameter, implicating the importance of blood flow rate. Radial diffusion distances in vessels significantly correlated to axon number as a hyperbolic function, showing a need to engineer high numbers of small vessels in parallel to improving axonal densities. In conclusion, Schwann cells and eGFP-MSCs influenced the regenerating microenvironment with lasting effect on axonal and blood vessel growth. OPF+ scaffolds in a complete transection model allowed for a detailed comparative, histologic analysis of the cellular architecture in response to each cell type and provided insight into physiologic characteristics that may support axon regeneration.
doi:10.1089/ten.tea.2013.0551
PMCID: PMC4229864  PMID: 24854680
12.  Invertible micellar polymer nanoassemblies target bone tumor cells but not normal osteoblast cells 
Future Science OA  2015;1(3):FSO16.
Aim:
To demonstrate the capability of the invertible micellar polymer nanoassemblies (IMAs) to deliver and release curcumin using the recently discovered mechanism of macromolecular inversion to treat bone tumor cells.
Materials & Methods:
The effect of IMA-mediated delivery of curcumin on osteosarcoma cell survival was investigated using MTS assays. To assess the effect of IMAs-delivered curcumin on osteosarcoma cell growth, fluorescence-activated cell sorting was performed. The uptake of micellar nanoassemblies was followed using confocal microscopy.
Results & Discussion:
IMAs-delivered curcumin is effective in blocking osteosarcoma cell growth. It decreases cell viability in human osteosarcoma (MG63, KHOS, and LM7) cells while having no effect on normal human osteoblast cells. It indicates that curcumin-loaded IMAs provide a unique delivery system targeted to osteosarcoma cells.
Osteosarcoma is the most common primary bone malignancy that predominantly affects children and adolescents. Curcumin, a principal substance in the Asian spice turmeric, has been shown to block osteosarcoma cell growth. The clinical development of curcumin has been hindered due to poor aqueous solubility and thus, bioavailability, restricting its use as a drug. In this study, in order to improve the bioavailability and efficacy of curcumin, the drug was loaded (solubilized) into invertible micellar polymer nanoassemblies made from amphiphilic invertible polymers.
doi:10.4155/fso.15.14
PMCID: PMC5137969  PMID: 28031891
bone tumor treatment; curcumin delivery; invertible polymer micelles; micellar nanoassemblies
13.  Implantation of cauda equina nerve roots through a biodegradable scaffold at the conus medullaris in rat 
Background context
Traumatic injuries occurring at the conus medullaris of the spinal cord cause both permanent damage to the central nervous system, and to the cauda equina nerve roots.
Purpose
This proof of concept study determined whether implanting the nerve roots into a biodegradable scaffold would improve regeneration after injury.
Study design/setting
All experimental work involving rats was performed according to approved guidelines by the Mayo Clinic Institutional Animal Care and Use Committee (IACUC). Surgical procedures were performed on 32 Sprague Dawley rats. Four ventral cauda equina nerve roots were re-implanted either directly into the ventral cord stump or through a poly(lactic-co-glycolic acid) (PLGA) scaffold. These experimental groups were compared to a control group in which the nerves were inserted into a muscle fascia barrier that was placed between the spinal cord and nerve roots. Animals were sacrificed at four weeks.
Methods
This work was funded by the authors' institution; Morton Cure Paralysis Fund; The Craig H. Neilsen Foundation; and NIBIB grant R01 EB 02390. There was no conflict of interest between the study funding and the conclusions drawn.
Results
There was no difference in motor neuron counts in the spinal cord rostral to the injury in all treatment groups, implying equal potential for regeneration into implanted nerve roots. One-way ANOVA testing, with Tukey's post-test, showed a statistically significant improvement in axon regeneration through the injury in the PLGA scaffold treatment group compared to the control (p<0.05, scaffold n=11, control n=11).
Conclusion
This pilot study demonstrated that a PLGA scaffold improved regeneration of axons into peripheral nerve roots. However, the number of regenerating axons observed was limited and did not lead to functional recovery. Future experiments will employ a different scaffold material and possible growth factors or enzymes to increase axon populations.
doi:10.1016/j.spinee.2014.01.059
PMCID: PMC4125550  PMID: 24509005
14.  Recurrent PAX3-MAML3 Fusion in Biphenotypic Sinonasal Sarcoma 
Nature genetics  2014;46(7):666-668.
Biphenotypic sinonasal sarcoma (SNS) is a newly described tumor of the nasal and paranasal areas. Herein, we report the novel recurring chromosomal translocation t(2;4)(q35;q31.1) in SNS. The translocation results in the formation of the fusion protein PAX3-MAML3, which is a potent transcriptional activator of PAX3 response elements. The SNS phenotype is characterized by aberrant expression of genes involved in neuroectodermal and myogenic differentiation, which closely simulates the developmental roles of PAX3.
doi:10.1038/ng.2989
PMCID: PMC4236026  PMID: 24859338
PAX3; MAML3; fusion gene; rearrangement; sinonasal sarcoma
15.  Biomechanical Evaluation of an Injectable and Biodegradable Copolymer P(PF-co-CL) in a Cadaveric Vertebral Body Defect Model 
Tissue Engineering. Part A  2014;20(5-6):1096-1102.
A novel biodegradable copolymer, poly(propylene fumarate-co-caprolactone) [P(PF-co-CL)], has been developed in our laboratory as an injectable scaffold for bone defect repair. In the current study, we evaluated the ability of P(PF-co-CL) to reconstitute the load-bearing capacity of vertebral bodies with lytic lesions. Forty vertebral bodies from four fresh-frozen cadaveric thoracolumbar spines were used for this study. They were randomly divided into four groups: intact vertebral body (intact control), simulated defect without treatment (negative control), defect treated with P(PF-co-CL) (copolymer group), and defect treated with poly(methyl methacrylate) (PMMA group). Simulated metastatic lytic defects were made by removing a central core of the trabecular bone in each vertebral body with an approximate volume of 25% through an access hole in the side of the vertebrae. Defects were then filled by injecting either P(PF-co-CL) or PMMA in situ crosslinkable formulations. After the spines were imaged with quantitative computerized tomography, single vertebral body segments were harvested for mechanical testing. Specimens were compressed until failure or to 25% reduction in body height and ultimate strength and elastic modulus of each specimen were then calculated from the force–displacement data. The average failure strength of the copolymer group was 1.83 times stronger than the untreated negative group and it closely matched the intact vertebral bodies (intact control). The PMMA-treated vertebrae, however, had a failure strength 1.64 times larger compared with the intact control. The elastic modulus followed the same trend. This modulus mismatch between PMMA-treated vertebrae and the host vertebrae could potentially induce a fracture cascade and degenerative changes in adjacent intervertebral discs. In contrast, P(PF-co-CL) restored the mechanical properties of the treated segments similar to the normal, intact, vertebrae. Therefore, P(PF-co-CL) may be a suitable alternative to PMMA for vertebroplasty treatment of vertebral bodies with lytic defects.
doi:10.1089/ten.tea.2013.0275
PMCID: PMC3938939  PMID: 24256208
16.  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.
doi:10.4103/1673-5374.143424
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
17.  ELECTRICALLY CONDUCTIVE SURFACE MODIFICATIONS OF THREE-DIMENSIONAL POLYPROPYLENE FUMARATE SCAFFOLDS 
Summary
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
18.  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.
doi:10.1016/j.jconrel.2008.11.023
PMCID: PMC3974410  PMID: 19105972
Drug delivery; Controlled release; Bone morphogenetic protein-2; Single photon emission computed; tomography; Scintillation probes; Micro-computed tomography
19.  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.
doi:10.1016/j.actbio.2011.10.001
PMCID: PMC3972821  PMID: 22019759
microsphere; poly-lactic co-glycolic acid; vascular endothelial growth factor; bioactivity; biodegradation; nerve guide
20.  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.
doi:10.1016/j.actbio.2011.12.031
PMCID: PMC3970912  PMID: 22277774
Hydrogel; Bone regeneration; Osteoblast; Rabbit marrow stromal cells
21.  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.
doi:10.1016/j.actbio.2012.09.019
PMCID: PMC3970914  PMID: 23022545
pH-responsive; Doxorubicin; Microgels; Chordoma; Oligo(polyethylene glycol) fumarate
22.  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.
doi:10.1021/bm100526a
PMCID: PMC3947846  PMID: 20942380
hydrogel; electrical; conductive; nerve; tissue regeneration
23.  Evaluation of Osteoconductive Scaffolds in the Canine Femoral Multi-Defect Model 
Tissue Engineering. Part A  2013;19(5-6):634-648.
Treatment of large segmental bone defects remains an unsolved clinical challenge, despite a wide array of existing bone graft materials. This project was designed to rapidly assess and compare promising biodegradable osteoconductive scaffolds for use in the systematic development of new bone regeneration methodologies that combine scaffolds, sources of osteogenic cells, and bioactive scaffold modifications. Promising biomaterials and scaffold fabrication methods were identified in laboratories at Rutgers, MIT, Integra Life Sciences, and Mayo Clinic. Scaffolds were fabricated from various materials, including poly(L-lactide-co-glycolide) (PLGA), poly(L-lactide-co-ɛ-caprolactone) (PLCL), tyrosine-derived polycarbonate (TyrPC), and poly(propylene fumarate) (PPF). Highly porous three-dimensional (3D) scaffolds were fabricated by 3D printing, laser stereolithography, or solvent casting followed by porogen leaching. The canine femoral multi-defect model was used to systematically compare scaffold performance and enable selection of the most promising substrate(s) on which to add cell sourcing options and bioactive surface modifications. Mineralized cancellous allograft (MCA) was used to provide a comparative reference to the current clinical standard for osteoconductive scaffolds. Percent bone volume within the defect was assessed 4 weeks after implantation using both MicroCT and limited histomorphometry. Bone formed at the periphery of all scaffolds with varying levels of radial ingrowth. MCA produced a rapid and advanced stage of bone formation and remodeling throughout the defect in 4 weeks, greatly exceeding the performance of all polymer scaffolds. Two scaffold constructs, TyrPCPL/TCP and PPF4SLA/HAPLGA Dip, proved to be significantly better than alternative PLGA and PLCL scaffolds, justifying further development. MCA remains the current standard for osteoconductive scaffolds.
doi:10.1089/ten.tea.2012.0289
PMCID: PMC3568967  PMID: 23215980
24.  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.
doi:10.1016/j.bone.2012.10.015
PMCID: PMC3513670  PMID: 23085085
Histone deacetylase; Osteocalcin-Cre; Osteoblast; Osteocyte; DNA damage
25.  In Vivo Transplantation of Autogenous Marrow-Derived Cells Following Rapid Intraoperative Magnetic Separation Based on Hyaluronan to Augment Bone Regeneration 
Tissue Engineering. Part A  2012;19(1-2):125-134.
Introduction
This project was designed to test the hypothesis that rapid intraoperative processing of bone marrow based on hyaluronan (HA) could be used to improve the outcome of local bone regeneration if the concentration and prevalence of marrow-derived connective tissue progenitors (CTPs) could be increased and nonprogenitors depleted before implantation.
Methods
HA was used as a marker for positive selection of marrow-derived CTPs using magnetic separation (MS) to obtain a population of HA-positive cells with an increased CTP prevalence. Mineralized cancellous allograft (MCA) was used as an osteoconductive carrier scaffold for loading of HA-positive cells. The canine femoral multidefect model was used and four cylindrical defects measuring 10 mm in diameter and 15 mm in length were grafted with MCA combined with unprocessed marrow or with MS processed marrow that was enriched in HA+ CTPs and depleted in red blood cells and nonprogenitors. Outcome was assessed at 4 weeks using quantitative 3D microcomputed tomography (micro-CT) analysis of bone formation and histomorphological assessment.
Results
Histomorphological assessment showed a significant increase in new bone formation and in the vascular sinus area in the MS-processed defects. Robust bone formation was found throughout the defect area in both groups (defects grafted with unprocessed marrow or with MS processed marrow.) Percent bone volume in the defects, as assessed by micro-CT, was greater in defects engrafted with MS processed cells, but the difference was not statistically significant.
Conclusion
Rapid intraoperative MS processing to enrich CTPs based on HA as a surface marker can be used to increase the concentration and prevalence of CTPs. MCA grafts supplemented with heparinized bone marrow or MS processed cells resulted in a robust and advanced stage of bone regeneration at 4 weeks. A greater new bone formation and vascular sinus area was found in defects grafted with MS processed cells. These data suggest that MS processing may be used to enhance the performance of marrow-derived CTPs in clinical bone regeneration procedures. Further assessment in a more stringent bone defect model is proposed.
doi:10.1089/ten.tea.2011.0622
PMCID: PMC3593694  PMID: 23082937

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