The effect of low-magnitude, high-frequency vibration on poly(ethylene glycol)-microencapsulated mesenchymal stem cells
Low-magnitude, high-frequency vibration has stimulated osteogenesis in mesenchymal stem cells when these cells were cultured in certain types of three-dimensional environments. However, results of osteogenesis are conflicting with some reports showing no effect of vibration at all. A large number of vibration studies using three-dimensional scaffolds employ scaffolds derived from natural sources. Since these natural sources potentially have inherent biochemical and microarchitectural cues, we explored the effect of low-magnitude, high-frequency vibration at low, medium, and high accelerations when mesenchymal stem cells were encapsulated in poly(ethylene glycol) diacrylate microspheres. Low and medium accelerations enhanced osteogenesis in mesenchymal stem cells while high accelerations inhibited it. These studies demonstrate that the isolated effect of vibration alone induces osteogenesis.
The influence of substrate modulus on retinal pigment epithelial cells
Although transplantation of retinal pigment epithelial (RPE) cells has shown promise for the treatment of retinal degenerative diseases, this therapeutic approach is not without challenges. Two major challenges are RPE cell dedifferentiation and inflammatory response following transplantation. The aim of this work is to understand how the rigidity of a scaffold, a relatively unexplored design aspect in retinal tissue engineering, affects RPE cells, particularly the pathways associated with the aforementioned challenges. Poly(ethylene glycol) diacrylate (PEGDA) of varying molecular weights from 3.4 to 20 kDa were photopolymerized to fabricate scaffolds. The Young’s modulus of the scaffolds varied from 60 to 1200 kPa. A cell study was then conducted to test the effects of scaffold rigidity on RPE cells. A cell adhesion peptide motif of arginine‐glycine‐aspartic acid‐serine (RGDS) was conjugated to 60 and 1200 kPa scaffolds and ARPE‐19 cells, a human RPE cell line, were seeded onto these hydrogels. Cells grown on scaffolds demonstrated qualitatively different adhesion properties, metabolic activity, and gene expression at an mRNA level. IL‐6 and MCP‐1, two inflammation markers known to recruit microglial into the retina, had the same expression pattern with cells having the highest expression on the high modulus scaffold and lowest expression on the control substrate. This study demonstrates that scaffold rigidity, an important design parameter in other areas of tissue engineering, affects cell adhesion, activity, and expression of RPE cells. Though more exploration is needed, this begins to lay a foundation for optimizing scaffold rigidity to promote long‐term success of RPE scaffolds. © 2017 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1260–1266, 2017.
The effects of botulinum toxin A on muscle histology during distraction osteogenesis
Distraction osteogenesis is a highly successful method of bone formation, yet muscle fibrosis and contractures can result in significant morbidity. In the current study, we investigate the efficacy of botulinum toxin A in preventing fibrosis and potentially increasing muscle development in distracted muscles. Fifteen New Zealand White rabbits underwent tibial distraction at 1.5 mm/day until a 20% gain was achieved. Treatment groups were divided by drug (saline or botulinum toxin) and target muscle (gastrocnemius or tibialis anterior). Two additional control animals received no treatment. Bromeodeoxyuridine was delivered continuously throughout the 8‐week experiment, and following muscle harvest. Tissues were stained for BrdU, Pax‐7, vimentin, and haematoxylin and eosin staining. Mitotic activity increased in all distracted animals; however, in the animals receiving botulinum toxin A injections into the gastrocnemius, the antagonist tibialis anterior suffered up to 9% less fibrosis than distraction alone (p = 0.024). Use of botulinum A toxin did not appear to promote or improve neogenesis of muscle fibers, nor did it decrease fibrosis in the injected muscles. It appears from this study, and a previously published study on the effects of this toxin on muscle function, that botulinum A toxin maybe of some benefit in decreasing morbidity in the antagonist muscle but not the muscle injected with the toxin. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:310–317, 2009
Scaffolds for retinal pigment epithelial cell transplantation in age-related macular degeneration
In several retinal degenerative diseases, including age-related macular degeneration, the retinal pigment epithelium, a highly functionalized cell monolayer, becomes dysfunctional. These retinal diseases are marked by early retinal pigment epithelium dysfunction reducing its ability to maintain a healthy retina, hence making the retinal pigment epithelium an attractive target for treatment. Cell therapies, including bolus cell injections, have been investigated with mixed results. Since bolus cell injection does not promote the proper monolayer architecture, scaffolds seeded with retinal pigment epithelium cells and then implanted have been increasingly investigated. Such cell-seeded scaffolds address both the dysfunction of the retinal pigment epithelium cells and age-related retinal changes that inhibit the efficacy of cell-only therapies. Currently, several groups are investigating retinal therapies using seeded cells from a number of cell sources on a variety of scaffolds, such as degradable, non-degradable, natural, and artificial substrates. This review describes the variety of scaffolds that have been developed for the implantation of retinal pigment epithelium cells.
Strain and Vibration in Mesenchymal Stem Cells
Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into any mesenchymal tissue, including bone, cartilage, muscle, and fat. MSC differentiation can be influenced by a variety of stimuli, including environmental and mechanical stimulation, scaffold physical properties, or applied loads. Numerous studies have evaluated the effects of vibration or cyclic tensile strain on MSCs towards developing a mechanically based method of differentiation, but there is no consensus between studies and each investigation uses different culture conditions, which also influence MSC fate. Here we present an overview of the response of MSCs to vibration and cyclic tension, focusing on the effect of various culture conditions and strain or vibration parameters. Our review reveals that scaffold type (e.g., natural versus synthetic; 2D versus 3D) can influence cell response to vibration and strain to the same degree as loading parameters. Hence, in the efforts to use mechanical loading as a reliable method to differentiate cells, scaffold selection is as important as method of loading.
Effects of botulinum toxin A on functional outcome during distraction osteogenesis
Distraction osteogenesis is useful for correcting limb length inequality, deformities, or short stature. Despite success with bone formation, soft tissue maladaptations including muscle and joint contracture may lead to undesirable results. Botulinum toxin A has been useful in treating spasticity in cerebral palsy, and has been used clinically in select cases to allay contracture in distraction osteogenesis. This study examines the toxin’s efficacy in preventing distraction‐induced loss of muscle strength and range of motion. The left tibias of 15 New Zealand White rabbits were distracted 1.5 mm/day until approximately a 20% gain was achieved. Each treatment group was divided into animals injected with saline or botulinum toxin in either the gastrocnemius or tibialis anterior muscles. A control group of two additional animals underwent no surgical procedure. Strength and range of motion were assessed prior to, and following, the experiment. At the study’s end, animals were euthanized and muscles were harvested, when lengths and weights were recorded. All muscles injected with botulinum toxin showed decreased wet weight and persistent weakness upon completion of the study. Range of motion decreased in all distracted animals. When the gastrocnemius was injected, its strength was reduced but the tibialis anterior strength was preserved, and the limb achieved 22% greater dorsiflexion than saline controls (p = 0.016). When the tibialis anterior received the toxin, plantarflexion was increased by 23% (p = 0.049). Botulinum toxin injection prior to limb distraction increases the “post‐lengthened” excursion of the injected muscle and this increased length may have a protective effect on its antagonist. In toxin‐injected gastrocnemius muscles, the level of equinus contracture is reduced due to length gains in the Achilles tendon while the anterior tibialis maintains its ability to generate torque. Injection of botulinum toxin in the gastrocnemius may minimize equinus contracture and protect the anterior tibialis from damage during human tibial lengthening. Longer follow‐up studies are needed to ensure that toxin‐induced muscle weakness resolves with time. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:656–664, 2007
Poly(3,4‐ethylenedioxythiophene) nanoparticle and poly(ɛ‐caprolactone) electrospun scaffold characterization for skeletal muscle regeneration
Injuries to peripheral nerves and/or skeletal muscle can cause scar tissue formation and loss of function. The focus of this article is the creation of a conductive, biocompatible scaffold with appropriate mechanical properties to regenerate skeletal muscle. Poly(3,4‐ethylenedioxythiophene) (PEDOT) nanoparticles (Np) were electrospun with poly(ɛ‐caprolactone) (PCL) to form conductive scaffolds. During electrospinning, ribboning, larger fiber diameters, and unaligned scaffolds were observed with increasing PEDOT amounts. To address this, PEDOT Np were sonicated prior to electrospinning, which resulted in decreased conductivity and increased mechanical properties. Multi‐walled carbon nanotubes (MWCNT) were added to the 1:2 solution in an effort to increase conductivity. However, the addition of MWCNT had little effect on scaffold conductivity, and the elastic modulus and yield stress of the scaffold increased as a result. Rat muscle cells attached and were active on the 1–10, 1–2, 3–4, and 1–1 PCL‐PEDOT scaffolds; however, the 3–4 scaffolds had the lowest level of metabolic activity. Although the scaffolds were cytocompatible, further development of the fabrication method is necessary to produce more highly aligned scaffolds capable of promoting skeletal muscle cell alignment and eventual regeneration. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 3633–3641, 2015.
An injectable method for noninvasive spine fusion
Bone morphogenetic proteins (BMPs) induce bone formation but are difficult to localize, and subsequent diffusion from the site of interest and short half-life reduce the efficacy of the protein. Currently, spine fusion requires stripping, decortications of the transverse processes, and an autograft harvest procedure. Even in combination with BMPs, clinical spinal fusion has a high failure rate, presumably because of difficulties in localizing sufficient levels of BMP.
The goal was to achieve reliable spine fusion through a single injection of a cell-based gene therapy system without the need for any surgical intervention.
Eighty-seven immunodeficient (n=44) and immune-competent (n=43) mice were injected along the paraspinous musculature to achieve rapid induction of heterotopic ossification (HO) and ultimately spinal arthrodesis.
Immunodeficient and immune-competent mice were injected with fibroblasts, transduced with an adenoviral vector to express BMP2, along the paraspinous musculature. Bone formation was evaluated via radiographs, microcomputed tomography, and biomechanical analysis.
ew bridging bone between the vertebrae and the fusion to adjacent skeletal bone was obtained as early as 2 weeks. Reduction in spine flexion-extension also occurred as early as 2 weeks after injection of the gene therapy system, with greater than 90% fusion by 4 weeks in all animals regardless of their genetic background.
Injection of our cell-based system into the paraspinous musculature induces spinal fusion that is dependent neither on the cell type nor on the immune status. These studies are the first to harness HO in an immune-competent model as a noninvasive injectable system for clinically relevant spinal fusion and may one day impact human spinal arthrodesis.
Rapid Heterotrophic Ossification with Cryopreserved Poly(ethylene glycol-) Microencapsulated BMP2-Expressing MSCs
Autologous bone grafting is the most effective treatment for long-bone nonunions, but it poses considerable risks to donors, necessitating the development of alternative therapeutics. Poly(ethylene glycol) (PEG) microencapsulation and BMP2 transgene delivery
are being developed together to induce rapid bone formation. However, methods to make these treatments available for clinical applications are presently lacking. In this study we used mesenchymal stem cells (MSCs) due to their ease of harvest, replication
potential, and immunomodulatory capabilities. MSCs were from sheep and pig due to their appeal as large animal models for
bone nonunion. We demonstrated that cryopreservation of these microencapsulated MSCs did not affect their cell viability, adenoviral BMP2 production, or ability to initiate bone formation. Additionally, microspheres showed no appreciable damage from cryopreservation when examined with light and electron microscopy. These results validate the use of cryopreservation in preserving
the viability and functionality of PEG-encapsulated BMP2-transduced MSCs.
Cell‐based gene therapy for repair of critical size defects in the rat fibula
More than a decade has passed since the first experiments using adenovirus‐transduced cells expressing bone morphogenetic protein 2 were performed for the synthesis of bone. Since this time, the field of bone gene therapy has tackled many issues surrounding safety and efficacy of this type of strategy. We present studies examining the parameters of the timing of bone healing, and remodeling when heterotopic ossification (HO) is used for bone fracture repair using an adenovirus gene therapy approach. We use a rat fibula defect, which surprisingly does not heal even when a simple fracture is introduced. In this model, the bone quickly resorbs most likely due to the non‐weight bearing nature of this bone in rodents. Using our gene therapy system robust HO can be introduced at the targeted location of the defect resulting in bone repair. The HO and resultant bone healing appeared to be dose dependent, based on the number of AdBMP2‐transduced cells delivered. Interestingly, the HO undergoes substantial remodeling, and assumes the size and shape of the missing segment of bone. However, in some instances we observed some additional bone associated with the repair, signifying that perhaps the forces on the newly forming bone are inadequate to dictate shape. In all cases, the HO appeared to fuse into the adjacent long bone. The data collectively indicates that the use of BMP2 gene therapy strategies may vary depending on the location and nature of the defect. Therefore, additional parameters should be considered when implementing such strategies. J. Cell. Biochem. 112: 1563–1571, 2011. © 2011 Wiley‐Liss, Inc.