Pegylated insulin-like growth factor-1 biotherapeutic delivery promotes rotator cuff regeneration in a rat model.

Tears in the rotator cuff are challenging to repair because of the complex, hypocellular, hypovascular, and movement-active nature of the tendon and its enthesis. Insulin-like Growth Factor-1 (IGF-1) is a promising therapeutic for this repair. However, its unstable nature, short half-life, and ability to disrupt homeostasis has limited its clinical translation. Pegylation has been shown to improve the stability and sustain IGF-1 levels in the systemic circulation without disrupting homeostasis. To provide localized delivery of IGF-1 in the repaired tendons, we encapsulated pegylated IGF-1 mimic and its controls (unpegylated IGF-1 mimic and recombinant human IGF-1) in polycaprolactone-based matrices and evaluated them in a pre-clinical rodent model of rotator cuff repair. Pegylated-IGF-1 mimic delivery reestablished the characteristic tendon-to-bone enthesis structure and improved tendon tensile properties within 8 weeks of repair compared to controls, signifying the importance of pegylation in this complex tissue regeneration. These results demonstrate a simple and scalable biologic delivery technology alternative to tissue-derived grafts for soft tissue repair.

Kinetic degradation and biocompatibility evaluation of polycaprolactone-based biologics delivery matrices for regenerative engineering of the rotator cuff.

Whereas synthetic biodegradable polymers have been successfully applied for the delivery of biologics in other tissues, the anatomical complexity, poor blood supply, and reduced clearance of degradation byproducts in the rotator cuff create unique design challenges for implantable biomaterials. Here, we investigated lower molecular weight poly-lactic acid co-epsilon-caprolactone (PLA-CL) formulations with varying molecular weight and film casting concentrations as potential matrices for the therapeutic delivery of biologics in the rotator cuff. Matrices were fabricated with target footprint dimensions to facilitate controlled and protected release of model biologic (Bovine Serum Albumin), and anatomically-unhindered implantation under the acromion in a rodent model of acute rotator cuff repair. The matrix obtained from the highest polymeric-film casting concentration showed a controlled release of model biologics payload. The tested matrices rapidly degraded during the initial 4 weeks due to preferential hydrolysis of the lactide-rich regions within the polymer, and subsequently maintained a stable molecular weight due to the emergence of highly-crystalline caprolactone-rich regions. pH evaluation in the interior of the matrix showed minimal change signifying lesser accumulation of acidic degradation byproducts than seen in other bulk-degrading polymers, and maintenance of conformational stability of the model biologic payload. The context-dependent biocompatibility evaluation in a rodent model of acute rotator cuff repair showed matrix remodeling without eliciting excessive inflammatory reaction and is anticipated to completely degrade within 6 months. The engineered PLA-CL matrices offer unique advantages in controlled and protected biologic delivery, non-toxic biodegradation, and biocompatibility overcoming several limitations of commonly-used biodegradable polyesters.

The Treatment of Muscle Atrophy after Rotator Cuff Tears Using Electroconductive Nanofibrous Matrices.

Rotator cuff tears (RCTs) are a common cause of disability and pain in the adult population. Despite the successful repair of the torn tendon, the delay between the time of injury and time of repair can cause muscle atrophy. The goal of the study was to engineer an electroconductive nanofibrous matrix with an aligned orientation to enhance muscle regeneration after rotator cuff (RC) repair. The electroconductive nanofibrous matrix was fabricated by coating Poly(3,4-ethylenedioxythiophene): poly(styrenesulfonate) (PEDOT:PSS) nanoparticles onto the aligned poly(ε-caprolactone) (PCL) electrospun nanofibers. The regenerative potential of the matrix was evaluated using two repair models of RCTs include acute and sub-acute. Sprague-Dawley rats (n=39) were randomly assigned to 1 of 8 groups. For the acute model, the matrix was implanted on supraspinatus muscle immediately after the injury. The repair surgery for the sub-acute model was conducted 6 weeks after injury. The supraspinatus muscle was harvested for histological analysis two and six weeks after repair. The results demonstrated the efficacy of electrical and topographical cues on the treatment of muscle atrophy in vivo. In both acute and sub-acute models, the stimulus effects of topographical and electrical cues reduced the gap area between muscle fibers. This study showed that muscle atrophy can be alleviated by successful surgical repair using an electroconductive nanofibrous matrix in a rat RC model.

3-D multi-electrode arrays detect early spontaneous electrophysiological activity in 3-D neuronal-astrocytic co-cultures.

Three-dimensional (3-D) neural cultures represent a promising platform for studying disease and drug screening. Tools and methodologies for measuring the electrophysiological function in these cultures are needed. Therefore, the purpose of this work was primarily to develop a methodology to interface engineered 3-D dissociated neural cultures with commercially available 3-D multi-electrode arrays (MEAs) reliably over 3 weeks to enable the recording of their electrophysiological activity. We further compared the functional output of these cultures to their structural and synaptic network development over time. We reliably interfaced a primary rodent neuron-astrocyte (2:1) 3-D co-culture (2500 cells/mm3 plating cell density) in Matrigel™ (7.5 mg/mL) that was up to 750 µm thick (30-40 cell-layers) with spiked 3-D MEAs while maintaining high viability. Using these MEAs we successfully recorded the spontaneous development of neural network-level electrophysiological activity and measured the development of putative synapses and neuronal maturation in these co-cultures using immunocytochemistry over 3 weeks in vitro. Planar (2-D) MEAs interfaced with these cultures served as recording controls. Neurons within this interfaced 3-D culture-MEA system exhibited considerable neurite outgrowth, networking, neuronal maturation, synaptogenesis, and culture-wide spontaneous firing of synchronized spikes and bursts of action potentials. Network-wide spikes and synchronized bursts increased rapidly (first detected at 2 days) during the first week in culture, plateaued during the second week, and reduced slightly in the third week, while maintaining high viability throughout the 3-week culturing period. Early electrophysiology activity occurred prior to neuronal process maturation and significant synaptic density increases in the second week. We successfully interfaced 3-D neural co-cultures with 3-D MEAs and recorded the electrophysiological activity of these cultures over 3 weeks. The initial period of rapid increase in electrophysiological activity, followed by a period of neuronal maturation and high-level of synapse formation in these cultures suggests a developmental homeostatic process. This methodology can enable future applications both in fundamental investigations of neural network behavior and in translational studies involving drug testing and neural interfacing.

Growth factor delivery strategies for rotator cuff repair and regeneration.

The high incidence of degenerative tears and prevalence of retears (20-95%) after surgical repair makes rotator cuff injuries a significant health problem. This high retear rate is attributed to the failure of the repaired tissue to regenerate the native tendon-to-bone insertion (enthesis). Biological augmentation of surgical repair such as autografts, allografts, and xenografts are confounded by donor site morbidity, immunogenicity, and disease transmission, respectively. In contrast, these risks may be alleviated via growth factor therapy, which can actively influence the healing environment to promote functional repair. Several challenges have to be overcome before growth factor delivery can translate into clinical practice such as the selection of optimal growth factor(s) or combination, identification of the most efficient stage and duration of delivery, and the design considerations for the delivery device. Emerging insight into the injury-repair microenvironment and our understanding of growth factor mechanisms in healing are informing the design of advanced delivery scaffolds to effectively treat rotator cuff tears. Here, we review potential growth factor candidates, design parameters and material selection for growth factor delivery, innovative and dynamic delivery scaffolds, and novel therapeutic targets from tendon and developmental biology for the structural and functional healing of rotator cuff repair.

A Device for Long-Term Perfusion, Imaging, and Electrical Interfacing of Brain Tissue In vitro.

Distributed microelectrode array (MEA) recordings from consistent, viable, ≥500 µm thick tissue preparations over time periods from days to weeks may aid in studying a wide range of problems in neurobiology that require in vivo-like organotypic morphology. Existing tools for electrically interfacing with organotypic slices do not address necrosis that inevitably occurs within thick slices with limited diffusion of nutrients and gas, and limited removal of waste. We developed an integrated device that enables long-term maintenance of thick, functionally active, brain tissue models using interstitial perfusion and distributed recordings from thick sections of explanted tissue on a perforated multi-electrode array. This novel device allows for automated culturing, in situ imaging, and extracellular multi-electrode interfacing with brain slices, 3-D cell cultures, and potentially other tissue culture models. The device is economical, easy to assemble, and integrable with standard electrophysiology tools. We found that convective perfusion through the culture thickness provided a functional benefit to the preparations as firing rates were generally higher in perfused cultures compared to their respective unperfused controls. This work is a step toward the development of integrated tools for days-long experiments with more consistent, healthier, thicker, and functionally more active tissue cultures with built-in distributed electrophysiological recording and stimulation functionality. The results may be useful for the study of normal processes, pathological conditions, and drug screening strategies currently hindered by the limitations of acute (a few hours long) brain slice preparations.

Poly (lactic acid)-based biomaterials for orthopaedic regenerative engineering.

Regenerative engineering converges tissue engineering, advanced materials science, stem cell science, and developmental biology to regenerate complex tissues such as whole limbs. Regenerative engineering scaffolds provide mechanical support and nanoscale control over architecture, topography, and biochemical cues to influence cellular outcome. In this regard, poly (lactic acid) (PLA)-based biomaterials may be considered as a gold standard for many orthopaedic regenerative engineering applications because of their versatility in fabrication, biodegradability, and compatibility with biomolecules and cells. Here we discuss recent developments in PLA-based biomaterials with respect to processability and current applications in the clinical and research settings for bone, ligament, meniscus, and cartilage regeneration.

Regenerative Engineering of Cartilage Using Adipose-Derived Stem Cells.

Injury to the articular cartilage occurs commonly in the general population and undergoes minimal spontaneous healing. Traditional methods of cartilage repair provide no long-term cure and are significant causes of morbidity. For this reason, stem cell therapies have recently been investigated for their ability to regenerate cartilage, and the results have been promising. Since the discovery that adipose tissue is a major source of mesenchymal stem cells in 2001, scientists have been studying the use of adipose-derived stem cells (ASCs) for the treatment of various disorders including lesions of the articular cartilage. ASCs hold several advantages over autologous chondrocytes for cartilage repair, including but not limited to their anti-inflammatory effects, their multi-lineage differentiation potential, and their ability to form new cartilage in a defect. Whereas several investigations have been made in in vitro and animal models, there have been surprisingly little clinical studies on the intra-articular use of adipose-derived stem cells, despite their first isolation about a decade and a half ago. The few studies that have been conducted are encouraging. With approval for various stem cell therapies on the horizon, this review seeks to update the clinician and the researcher on the current state-of-the-art use of adipose-derived stem cells for the treatment of cartilage disorders and the regenerative engineering of cartilaginous tissue.

Bi-ligand surfaces with oriented and patterned protein for real-time tracking of cell migration.

A bioactive platform for the quantitative observation of cell migration is presented by (1) presenting migration factors in a well-defined manner on 2-D substrates, and (2) enabling continuous cell tracking. Well-defined substrate presentation is achieved by correctly orienting immobilized proteins (chemokines and cell adhesion molecules), such that the active site is accessible to cell surface receptors. A thiol-terminated self-assembled monolayer on a silica slide was used as a base substrate for subsequent chemistry. The thiol-terminated surface was converted to an immobilized metal ion surface using a maleimido-nitrilotriacetic acid (NTA) cross-linker that bound Histidine-tagged recombinant proteins on the surface with uniform distribution and specific orientation. This platform was used to study the influence of surface-immobilized chemokine SDF-1α and cell adhesion molecule ICAM-1 on murine splenic B lymphocyte migration. While soluble SDF-1α induced trans-migration in a Boyden Chamber type chemotaxis assay, immobilized SDF-1α alone did not elicit significant surface-migration on our test-platform surface. Surface-immobilized cell adhesion protein, ICAM-1, in conjunction with activation enabled migration of this cell type on our surface. Controlled exposure to UV light was used to produce stable linear gradients of His-tagged recombinant SDF-1α co-immobilized with ICAM-1 following our surface chemistry approach. XPS and antibody staining showed defined gradients of outwardly oriented SDF-1α active sites. This test platform can be especially valuable for investigators interested in studying the influence of surface-immobilized factors on cell behavior and may also be used as a cell migration enabling platform for testing the effects of various diffusible agents.

Trauma-induced plasmalemma disruptions in three-dimensional neural cultures are dependent on strain modality and rate.

Traumatic brain injury (TBI) results from cell dysfunction or death following supra-threshold physical loading. Neural plasmalemma compromise has been observed following traumatic neural insults; however, the biomechanical thresholds and time-course of such disruptions remain poorly understood. In order to investigate trauma-induced membrane disruptions, we induced dynamic strain fields (0.50 shear or compressive strain at 1, 10, or 30?sec(?1) strain rate) in 3-D neuronal-astrocytic co-cultures (>500??m thick). Impermeant dyes were present during mechanical loading and entered cells in a strain rate-dependent manner for both shear and compression. Real-time imaging revealed increased membrane permeability in a sub-population of cells immediately upon deformation. Alterations in cell membrane permeability, however, were transient and biphasic over the ensuing hour post-insult, suggesting initial membrane damage and rapid repair, followed by a phase of secondary membrane degradation. At 48?h post-insult, cell death increased significantly in the high-strain-rate group, but not after quasi-static loading, suggesting that cell survival relates to the initial extent of transient structural compromise. Cells were more sensitive to bulk shear deformation than compression with respect to acute permeability changes and subsequent cell survival. These results provide insight into the temporally varying alterations in membrane stability following traumatic loading and provide a basis for elucidating physical cellular tolerances.

Neural tissue engineering and biohybridized microsystems for neurobiological investigation in vitro (Part 1).

Advances in neural tissue engineering have resulted in the development and implementation of three-dimensional (3-D) neural cellular constructs, which may serve as neurofidelic in vitro investigational platforms. In addition, interfacing these 3-D cellular constructs with micro-fluidic and/or micro-electrical systems has created biohybridized platforms, providing unprecedented 3-D microenvironmental control and allowing noninvasive probing and manipulation of cultured neural cells. Cells in the brain interact within a complex, multicellular environment with tightly coupled 3-D cell-cell/cell-extracellular matrix (ECM) interactions; yet most in vitro models utilize planar systems lacking in vivo-like ECM. As such, neural cultures with cells distributed throughout a thick (> 500 microm), bioactive extracellular matrix may provide a more physiologically relevant setting to study neurobiological phenomena than traditional planar cultures. This review presents an overview of 2-D versus 3-D culture models and the state of the art in 3-D neural cell-culture systems. We then detail our efforts to engineer a range of 3-D neural cellular constructs by systematically varying parameters such as cell composition, cell density, matrix constituents, and mass transport. The ramifications on neural cell survival, function, and network formation based on these parameters are specifically addressed. These 3-D neural cellular constructs may serve as powerful investigational platforms for the study of basic neurobiology, network neurophysiology, injury/disease mechanisms, pharmacological screening, or test-beds for cell replacement therapies. Furthermore, while survival and growth of neural cells within 3-D constructs poses many challenges, optimizing in vitro constructs prior to in vivo implementation offers a sound bioengineering design approach.

SU-8 2000 rendered cytocompatible for neuronal bioMEMS applications.

Microfabrication advances have resulted in small, cheap, and precise devices for biological microelectromechanical systems (bioMEMS). SU-8/SU-8 2000 is an attractive material for these applications because of its high-aspect ratio fabrication capability, dielectric properties, and thermochemical stability. Despite these advantages, the potential toxicity of SU-8 2000 may limit its use in cell-based applications. We show that <10% of primary neurons survived when cultured adjacent to or on top of untreated SU-8 2000. We evaluated the efficacy of various detoxification and surface treatments for SU-8 2000 in neuronal cultures after 7-21 days in vitro. Viability was improved to 45.8% +/- 4.5% (mean +/- standard error of the mean) following 3-day heat treatment (150 degrees C) under vacuum, while UV exposure and CO2 supercritical extraction did not improve survival. Furthermore, parylene coating (25 microm), in combination with heat and sonication (in isopropanol) treatments effectively masked the SU-8 2000 and led to 86.4% +/- 1.9% viability. Glow discharge (oxygen plasma) treatment rendered the SU-8 2000 surface more hydrophilic and improved neuronal viability, possibly through improved cell adhesion. No organic leachants were detected by mass spectrometry before or after heat treatment or after sonication. However, XPS analysis revealed the presence of potentially neurotoxic elements, fluorine and antimony. Strategies to improve the cytocompatibility of SU-8 2000 with primary neurons will allow longer culture times and have applications for cell-based microfabrication.