Instructive Conductive 3D Silk Foam-Based Bone Tissue Scaffolds Enable Electrical Stimulation of Stem Cells for Enhanced Osteogenic Differentiation.

Stimuli-responsive materials enabling the behavior of the cells that reside within them to be controlled are vital for the development of instructive tissue scaffolds for tissue engineering. Herein, we describe the preparation of conductive silk foam-based bone tissue scaffolds that enable the electrical stimulation of human mesenchymal stem cells (HMSCs) to enhance their differentiation toward osteogenic outcomes.

Cell-tethered ligands modulate bone remodeling by osteoblasts and osteoclasts.

The goals of the present study are to establish an in vitro co-culture model of osteoblast and osteoclast function and to quantify the resulting bone remodeling. The bone is tissue engineered using well-defined silk protein biomaterials in 2D and 3D formats in combination with human cells expressing tethered agonists for selected G protein-coupled receptors (GPCRs). The tethered constructs are introduced with the objective of triggering sustained and localized GPCR signaling. The cell-modified biomaterial surfaces are reconstructed from SEM images into 3D models using image processing for quantitative measurement of surface characteristics. Parathyroid hormone (PTH) and glucose-dependent insulinotropic peptide (GIP) are selected because of their roles in bone remodeling for expression in tethered format on bone marrow derived human mesenchymal stem cells (hMSCs). Increased calcium deposition and increased surface roughness are found in 3D digital surface models constructed from SEM images of silk protein films remodeled by the co-cultures containing the tethered PTH, and decreased surface roughness is found for the films remodeled by the tethered GIP co-cultures. Increased surface roughness is not found in monocultures of hMSCs expressing tethered PTH, suggesting that osteoclast-osteoblast interactions in the presence of PTH signaling are responsible for the increased mineralization. These data point towards the design of in vitro bone models in which osteoblast-osteoclast interactions are mimicked for a better understanding of bone remodeling.

Quantitative characterization of mineralized silk film remodeling during long-term osteoblast-osteoclast co-culture.

The goal of this study was to explore quantitative assessments of mineralized silk protein biomaterial films by co-cultures of human mesenchymal stem cell-derived osteoblasts and human acute monocytic leukemia cell line-derived osteoclasts during long-term culture (8-32 weeks). The remodeled films were quantitatively assessed using three different techniques during this extended cultivation to provide more comprehensive insight into the impact of co-cultures on surface remodeling. Scanning electron microscopy (SEM) with three dimensional surface reconstructions was used to quantitatively determine various surface morphological features and measures of roughness indicative of remodeling by the cells. Additionally, reconstructed surfaces were converted to depth images for Fourier analysis to quantify the potential fractal organization of biomineralization. The long-term remodeled films were also imaged using confocal reflectance microscopy and micro-computed tomography (micro-CT) to further quantify morphological changes. Films remodeled in co-culture demonstrated increased roughness parameters, fractal organization, and volume compared to films remodeled by osteoblasts alone. The combination of these techniques to quantify remodeling of mineralized protein films shows promise for quantifying processes related to mineralized surfaces.

Effects of clodronate and alendronate on osteoclast and osteoblast co-cultures on silk-hydroxyapatite films.

The goal of this study was to explore the effects of osteoporosis-related therapeutics on bone remodeling in vitro. A previously established bone-tissue mimetic system consisting of silk protein biomaterials in combination with hydroxyapatite and human cells was used for the study. Silk-hydroxyapatite films were pre-complexed with the non-nitrogenous bisphosphonate clodronate or the nitrogenous bisphosphonate alendronate and cultured with THP-1 human acute monocytic leukemia cell line-derived osteoclasts, human mesenchymal stem cell derived osteoblasts or a direct co-culture of the two cell types. Metabolic activity, calcium deposition and alkaline phosphatase activity were assessed over 12 weeks, and reconstructed remodeled biomaterial surfaces were also evaluated for quantitative morphological changes. Increased metabolic activity and increased roughness were found on the clodronate-complexed biomaterial substrates remodeled by osteoblasts and co-cultures of osteoblasts with osteoclasts, even at doses high enough to cause a 90% decrease in osteoclast metabolic activity. Films complexed with low doses of alendronate resulted in increased metabolic activity and calcium deposition by osteoblasts, while higher doses were similarly toxic among osteoclasts, osteoblasts and co-cultures. These results point to the utility of these well-defined bone-mimetic in vitro cultures as useful screens for therapeutics for bone-related diseases, particularly with the ability to conduct studies for extended duration (here for 12 weeks) and with pre-complexed drugs to mimic conditions found in vivo.

Quantitative metabolic imaging using endogenous fluorescence to detect stem cell differentiation.

The non-invasive high-resolution spatial mapping of cell metabolism within tissues could provide substantial advancements in assessing the efficacy of stem cell therapy and understanding tissue development. Here, using two-photon excited fluorescence microscopy, we elucidate the relationships among endogenous cell fluorescence, cell redox state, and the differentiation of human mesenchymal stem cells into adipogenic and osteoblastic lineages. Using liquid chromatography/mass spectrometry and quantitative PCR, we evaluate the sensitivity of an optical redox ratio of FAD/(NADH + FAD) to metabolic changes associated with stem cell differentiation. Furthermore, we probe the underlying physiological mechanisms, which relate a decrease in the redox ratio to the onset of differentiation. Because traditional assessments of stem cells and engineered tissues are destructive, time consuming, and logistically intensive, the development and validation of a non-invasive, label-free approach to defining the spatiotemporal patterns of cell differentiation can offer a powerful tool for rapid, high-content characterization of cell and tissue cultures.

Accelerated In Vitro Degradation of Optically Clear Low ß-Sheet Silk Films by Enzyme-Mediated Pretreatment.

PURPOSE: To design patterned, transparent silk films with fast degradation rates for the purpose of tissue engineering corneal stroma. METHODS: ß-sheet (crystalline) content of silk films was decreased significantly by using a short water annealing time. Additionally, a protocol combining short water annealing time with enzymatic pretreatment of silk films with protease XIV was developed. RESULTS: Low ß-sheet content (17%-18%) and enzymatic pretreatment provided film stability in aqueous environments and accelerated degradation of the silk films in the presence of human corneal fibroblasts in vitro. The results demonstrate a direct relationship between reduced ß-sheet content and enzymatic pretreatment, and overall degradation rate of the protein films. CONCLUSIONS: The novel protocol developed here provides new approaches to modulate the regeneration rate of silk biomaterials for corneal tissue regeneration needs. TRANSLATIONAL RELEVANCE: Patterned silk protein films possess desirable characteristics for corneal tissue engineering, including optical transparency, biocompatibility, cell alignment, and tunable mechanical properties, but current fabrication protocols do not provide adequate degradation rates to match the regeneration properties of the human cornea. This novel processing protocol makes silk films more suitable for the construction of human corneal stroma tissue and a promising way to tune silk film degradation properties to match corneal tissue regeneration.

A pilot study exploring the role of physical therapists and transition in care of pediatric patients with cystic fibrosis to the adult setting.

BACKGROUND: Cystic fibrosis (CF) is a disease that requires intensive multidisciplinary care, including care by physical therapists (PTs). People with CF are now living well into adulthood, necessitating a transfer of care from the pediatric setting to an adult one. Physical therapists play a large role in the care of the person with CF; however, there is little known about the PT role in transition of care. PURPOSE: To explore transition issues for people with CF from the perspective of PTs. METHODS: An 18-question online survey was sent to PTs via an electronic CF listserv. Questions were derived from an analysis of transition literature in CF and other chronic childhood conditions. Physical therapists who reported treating people with CF gave their opinions on issues impacting transition from their perspective as well as their perception of patient and parent concerns. Descriptive statistics were used for data analysis. RESULTS: A total of 26 PTs completed the survey. A majority, 61.5%, reported that there was a transition program at their facility, with 42.3% involving physical therapy. Common themes for patients and parents included feeling uncertain about: knowledge of the adult physician, acquiring pulmonary infections in the adult setting, and pace of the adult clinic. Physical therapists were concerned about adherence with airway clearance and exercise following transfer to the adult clinic. CONCLUSIONS: The role of PT in transition programs is quite varied. Physical therapists should address common concerns of their patients and families to improve the transition process and possibly impact adherence to the PT plan of care.

Multiple silk coatings on biphasic calcium phosphate scaffolds: effect on physical and mechanical properties and in vitro osteogenic response of human mesenchymal stem cells.

Ceramic scaffolds such as biphasic calcium phosphate (BCP) have been widely studied and used for bone regeneration, but their brittleness and low mechanical strength are major drawbacks. We report the first systematic study on the effect of silk coating in improving the mechanical and biological properties of BCP scaffolds, including (1) optimization of the silk coating process by investigating multiple coatings, and (2) in vitro evaluation of the osteogenic response of human mesenchymal stem cells (hMSCs) on the coated scaffolds. Our results show that multiple silk coatings on BCP ceramic scaffolds can achieve a significant coating effect to approach the mechanical properties of native bone tissue and positively influence osteogenesis by hMSCs over an extended period. The silk coating method developed in this study represents a simple yet effective means of reinforcement that can be applied to other types of ceramic scaffolds with similar microstructure to improve osteogenic outcomes.

The effect of rhythmic auditory stimulation (RAS) on physical therapy outcomes for patients in gait training following stroke: a feasibility study.

Adults aged 55 to 80 years participated voluntarily in a wait-list control study during in-patient physical therapy following first stroke. All participants (N = 15) received conventional physical therapy gait training throughout 30 treatment sessions. Rhythmic auditory stimulation (RAS)-enhanced gait training was nested within conventional treatments in three conditions: (1) RAS throughout 30 treatments (N = 5); (2) RAS in the last 20 treatments (N = 5); and (3) RAS in the last 10 treatments (N = 5). Cadence and balance outcome measurements were taken at baseline, and following 10, 20, and 30 treatment sessions. Improvements across time were statistically significant in all conditions for one-limb stance, cadence, velocity, stride length, and posture head tilt with no statistically significant improvements for the Timed Up and Go Test and the Functional Reach Test. Statistically significant gains were made in the one-limb stance and cadence with earlier implementations of RAS. Results of the study demonstrate the feasibility of RAS to enhance gait training that warrants further investigation of the protocol to demonstrate the effects of RAS in stroke rehabilitation.