Search

Silk fibroin interactions with metallic surfaces can provide utility for medical materials and devices. Toward this goal, titanium alloy (Ti6Al4 V) was covalently grafted with polyacrylamide via electrochemically reducing 4-nitrobenzene diazonium salt in the presence of acrylamide. Analysis of the modified surfaces with FT-IR spectra, SEM and AFM were consistent with surface grafting. Functionalised titanium samples with a silk fibroin membrane, with and without impregnated therapeutics, were used to assess cytocompatibility and drug delivery. Initial cytocompatibility experiments using fibroblasts showed that the functionalised samples, both with and without silk fibroin coatings, supported significant increases between 72-136 % in cell metabolism, compared to the controls after 7 days. A 7-days release profiling showed consistent bacterial inhibition through gentamicin release with average inhibition zones of 239 mm 2 . Over a 5-week period, silk fibroin coated samples, both with and without growth factors, supported better human mesenchymal stem cell metabolism with increases reaching 1031 % and 388 %, respectively, compared to samples without the silk fibroin coating with., (© 2023 The Authors. ChemPlusChem published by Wiley-VCH GmbH.)

Mesenchymal stem cells (MSCs) are widely used in regenerative medicine and tissue engineering and delivering biological molecules into MSCs has been used to control stem cell behavior. However, the efficient delivery of large biomolecules such as DNA, RNA, and proteins into MSCs using nonviral delivery strategies remains an ongoing challenge. Herein, nanoparticles composed of cationic bioreducible lipid-like materials (lipidoids) are developed to intracellularly deliver mRNA into human mesenchymal stem cells (hMSCs). The delivery efficacy to hMSCs is improved by adding three excipients including cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG) during lipidoid nanoparticle formulation. Using an optimized lipidoid formulation, Cas9 mRNA and single guide RNA (sgRNA) targeting neuron restrictive silencing factor (NRSF) are delivered to hMSCs, leading to successful neural-like differentiation as demonstrated by the expression of synaptophysin (SYP), brain-derived neurotrophic factor (BDNF), neuron-specific enolase (NSE), and neuron-specific growth-associated protein (SCG10). Overall, a synthetic lipid formulation that can efficiently deliver mRNA to hMSCs is identified, leading to CRISPR-based gene knockdown to facilitate hMSCs transdifferentiation into neural-like lineage., (© 2020 Wiley-VCH GmbH.)

Biomaterial scaffold designs are needed for self-organizing features related to tissue formation while also simplifying the fabrication processes involved. Toward this goal, silk protein-based self-folding scaffolds to support 3D cell culture, while providing directional guidance and promotion of cell growth and differentiation, are reported. A simple and robust one-step self-folding approach is developed using bilayers consisting of a hydrogel and silk film in aqueous solution. The 3D silk rolls, with patterns transferred from the initially prepared 2D films, guide the directional outgrowth of neurites and also promote the osteogenic differentiation of human mesenchymal stem cells (hMSCs). The osteogenic outcomes are further supported by enhanced biomechanical performance. By utilizing this self-folding method, cocultures of neurons and hMSCs are achieved by patterning cells on silk films and then converting these materials into a 3D format with rolling, mimicking aspects of the structure of osteons and providing physiologically relevant structures to promote bone regeneration. These results demonstrate the utility of self-folded silk rolls as efficient scaffold systems for tissue regeneration, while exploiting relatively simple 2D designs programmed to form more complex 3D structures., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

Scarless skin regeneration with functional tissue remains a challenge for full-thickness wounds. Here, mesenchymal stem cell (MSC)-laden hydrogels are developed for scarless wound healing with hair follicles. Microgels composed of aligned silk nanofibers are used to load MSCs to modulate the paracrine. MSC-laden microgels are dispersed into injectable silk nanofiber hydrogels, forming composites biomaterials containing the cells. The injectable hydrogels protect and stabilize the MSCs in the wounds. The synergistic action of silk-based composite hydrogels and MSCs stimulated angiogenesis and M1-M2 phenotype switching of macrophages, provides a suitable niche for functional recovery of wounds. Compared to skin defects treated with MSC-free hydrogels, the defects treated with the MSC-laden composite hydrogels heal faster and form scarless tissues with hair follicles. Wound healing can be further improved by adjusting the ratio of silk nanofibers and particles and the loaded MSCs, suggesting tunability of the system. To the best of current knowledge, this is the first time scarless skin regeneration with hair follicles based on silk material systems is reported. The improved wound healing capacity of the systems suggests future in vivo studies to compare to other biomaterial systems related to clinical goals in skin regeneration in the absence of scarring., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Gradient biomaterials are considered as preferable matrices for tissue engineering due to better simulation of native tissues. The introduction of gradient cues usually needs special equipment and complex process but is only effective to limited biomaterials. Incorporation of multiple gradients in the hydrogels remains challenges. Here, beta-sheet rich silk nanofibers (BSNF) were used as building blocks to introduce multiple gradients into different hydrogel systems through the joint action of crosslinking and electric field. The blocks migrated to the anode along the electric field and gradually stagnated due to the solution-hydrogel transition of the systems, finally achieving gradient distribution of the blocks in the formed hydrogels. The gradient distribution of the blocks could be tuned easily through changing different factors such as solution viscosity, which resulted in highly tunable gradient of mechanical cues. The blocks were also aligned under the electric field, endowing orientation gradient simultaneously. Different cargos could be loaded on the blocks and form gradient cues through the same crosslinking-electric field strategy. The building blocks could be introduced to various hydrogels such as Gelatin and NIPAM, indicating the universality. Complex niches with multiple gradient cues could be achieved through the strategy. Silk-based hydrogels with suitable mechanical gradients were fabricated to control the osteogenesis and chondrogenesis. Chondrogenic-osteogenic gradient transition was obtained, which stimulated the ectopic osteochondral tissue regeneration in vivo. The versatility and highly controllability of the strategy as well as multifunction of the building blocks reveal the applicability in complex tissue engineering and various interfacial tissues.

Silk-based bioresorbable medical devices, such as screws, plates, and rods, have been under investigation due to their promising properties for orthopedic repairs. Options to functionalize these new devices for enhanced control of bone regeneration would also exploit the compatible processing methods used to generate the devices. MicroRNAs are important regulators of bone maintenance and formation, and miRNA-based therapeutics have the potential to aid bone repair, utilizing a transient therapeutic approach with local bioactivity. We hypothesized that silk-based orthopedic devices could be used for the local delivery of miRNAs, using anti-sense miR-214 (AS-miR-214), to inhibit endogenous expression of osteoinductive antagonist and thereby supporting the upregulation of osteoinductive target molecules activating transcription factor 4 (ATF4) and Osterix (Osx). AS-miR-214 silk devices, prepared using surface coating, demonstrated continuous release of miRNA inhibitors up to 7 days in vitro. Additionally, human mesenchymal stem cells seeded on AS-miR-214 silk films expressed higher levels of osteogenic genes ATF4, Osx, Runx2, and Osteocalcin. Interestingly, these cells exhibited lower cell viability and DNA content over 21 days. Conversely, the cells demonstrated significantly higher levels of alkaline phosphatase expression and calcium deposition compared with cells seeded on silk films with nontargeting miRNA controls. The study demonstrated that the silk-based orthopedic devices, in conjunction with bioactive miRNA-based therapeutics, may serve as a novel system for localized bone tissue engineering, enhancing osteogenesis at the implant interface while avoiding detrimental systematic side effects.

Silk/polyethylene glycol (PEG) hydrogels are studied as self-standing bioinks for 3D printing for tissue engineering. The two components of the bioink, silk fibroin protein (silk) and PEG, are both Food and Drug Administration approved materials in drug and medical device products. Mixing PEG with silk induces silk β-sheet structure formation and thus gelation and water insolubility due to physical crosslinking. A variety of constructs with high resolution, high shape fidelity, and homogeneous gel matrices are printed. When human bone marrow mesenchymal stem cells are premixed with the silk solution prior to printing and the constructs are cultured in this medium, the cell-loaded constructs maintain their shape over at least 12 weeks. Interestingly, the cells grow faster in the higher silk concentration (10%, w/v) gel than in lower ones (7.5 and 5%, w/v), likely due to the difference in material stiffness and the amount of residual PEG remaining in the gel related to material hydrophobicity. Subcutaneous implantation of 7.5% (w/v) bioink gels with and without printed fibroblast cells in mice reveals that the cells survive and proliferate in the gel matrix for at least 6 week postimplantation. The results suggest that these silk/PEG bioink gels may provide suitable scaffold environments for cell printing and function., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The response of human bone marrow-derived mesenchymal stem cells (hMSCs) encapsulated in three-dimensional (3D) charged protein hydrogels was studied. Combining silk fibroin (S) with recombinant human tropoelastin (E) or silk ionomers (I) provided protein composite alloys with tunable physicochemical and biological features for regulating the bioactivity of encapsulated hMSCs. The effects of the biomaterial charges on hMSC viability, proliferation and chondrogenic or osteogenic differentiation were assessed. The silk-tropoelastin or silk-ionomers hydrogels supported hMSC viability, proliferation and differentiation. Gene expression of markers for chondrogenesis and osteogenesis, as well as biochemical and histological analysis, showed that hydrogels with different S/E and S/I ratios had different effects on cell fate. The negatively charged hydrogels upregulated hMSC chondrogenesis or osteogenesis, with or without specific differentiation media, and hydrogels with higher tropoelastin content inhibited the differentiation potential even in the presence of the differentiation media. The results provide insight on charge-tunable features of protein-based biomaterials to control hMSC differentiation in 3D hydrogels, as well as providing a new set of hydrogels for the compatible encapsulation and utility for cell functions. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Detailed control of nano- and microstructures in porous biomaterial scaffold systems is important for control of interfacial and biological functions. Self-assembled silk protein nanostructured building blocks were incorporated into salt-leached scaffolds to control these features. Controllable concentration and pH were used to induce the formation of amorphous silk nanofibers in solution and also to reduce β-sheet transformation during the more traditional salt-leaching process. These new scaffolds showed nanofibrous-microporous structures, reduced β-sheet content, and tunable mechanical properties. Bone marrow mesenchymal stem cells grew better and showed differentiation behavior on these nanofibrous scaffolds, suggesting cytocompatibility and support for tunable differentiation via the scaffolds. These results suggested a new strategy of designing bioactive silk scaffolds by combining traditional scaffold formation processes with the controllable self-assembly of silk.

Obesity is a rising issue especially in the United States that can lead to heart problems, type II diabetes, and respiratory problems. Since the 1970s, obesity rates in the United States have more than doubled in adults and children. Recent evidence suggests that exposure to certain chemicals, termed "obesogens," in utero may alter metabolic processes, predisposing individuals to weight gain. There is a need to develop a three-dimensional human tissue system that is able to model the effects of obesogens in vitro in order to better understand the impact of obesogens on early development. Human embryonic-derived stem cells in three-dimensional collagen embedded silk scaffolds were exposed to three different obesogens: Bisphenol A (BPA), Bisphenol S (BPS), and Tributyltin (TBT). The exposed tissues accumulated triglycerides and increased expression of adipogenic genes (Perilipin (PLIN1), peroxisome proliferator-activated receptor gamma (PPARy), fatty acid binding protein 4 (FABP4)) compared to equivalent control cultures with no obesogen exposure. These cultures were also compared to human adult stem cell cultures, which did not respond the same upon addition of obesogens. These results demonstrate the successful development of a representative tissue model of in utero obesogen exposures. This tissue system could be used to determine mechanisms of action of current obesogens and to screen other potential obesogens.

Bioactive ceramic scaffolds represent competitive choices for clinical bone reconstruction, but their widespread use is restricted by inherent brittleness and weak mechanical performance under load. This study reports the development of strong and tough bioactive scaffolds suitable for use in load-bearing bone reconstruction. A strong and bioactive ceramic scaffold (strontium-hardystonite-gahnite) is combined with single and multiple coating layers of silk fibroin to enhance its toughness, producing composite scaffolds which match the mechanical properties of cancellous bone and show enhanced capacity to promote in vitro osteogenesis. Also reported for the first time is a comparison of the coating effects obtained when a polymeric material is coated on ceramic scaffolds with differing microstructures, namely the strontium-hardystonite-gahnite scaffold with high-density struts as opposed to a conventional ceramic scaffold, such as biphasic calcium phosphate, with low-density struts. The results show that silk coating on a unique ceramic scaffold can lead to simple and effective enhancement of its mechanical and biological properties to suit a wider range of applications in clinical bone reconstruction, and also establish the influence of ceramic microstructure on the effectiveness of silk coating as a method of reinforcement when applied to different types of ceramic bone graft substitutes. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

Bone marrow aspirates (BMAs), owing to their innate osteogenic potential, are well-documented supplements to osteoconductive and/or osteoinductive materials. The calcaneal body provides foot and ankle surgeons a convenient harvest site with low morbidity and minimal cost. In the present study, we sought to identify and characterize multipotent mesenchymal stromal cells (MSCs) in BMAs harvested from the human calcaneal body. Ten healthy patients aged 18 to 65 years were enrolled in the present study. BMAs were harvested from the patients without any reported postoperative complications related to the harvest. Cells isolated from all the aspirates were adherent to culture plates and expressed positive MSC surface markers (CD105, CD90, and CD73) and a low level of negative MSC markers (CD34 and CD45). The cells maintained the ability to proliferate and differentiate into cells of mesenchymal lineages. The BMAs from the human calcaneal body offer a healthy source of multipotent MSCs., (Copyright © 2016 American College of Foot and Ankle Surgeons. Published by Elsevier Inc. All rights reserved.)

To produce biocompatible, mechanically robust, and conductive materials for bone tissue engineering, chemical oxidation using sodium hyprochlorite (NaClO) was utilized to introduce carboxyl groups onto silk fibroin (SF). A final carboxyl content of 1.09 mM/g SF was obtained, corresponding to ∼47% of the primary hydroxymethyl groups on the silk. Interestingly, both infrared (IR) spectroscopy and circular dichroism (CD) spectra demonstrated that the resulting oxidized silk (OxSF) self-assembled into β-sheet structures under aqueous conditions and this contributed to the mechanical properties of the as-prepared silk-based scaffolds and the mineralized OxSF scaffolds (M-OxSF). The OxSF scaffolds had a compressive modulus of 211 ± 75 KPa in the hydrated state, 10 times higher than that of the SF scaffolds, and the modulus of the M-OxSF scaffolds was increased to 758 ± 189 KPa. Human bone marrow-derived mesenchymal stem cells (hMSCs) grown on the scaffolds during osteogenesis showed that the OxSF scaffolds supported the proliferation and differentiation of hMSCs in vitro.

Bioelectrical regulation of bone fracture healing is important for many cellular events such as proliferation, migration, and differentiation. The aim of this study was to investigate the osteogenic differentiation potential of human mesenchymal stem cells (hMSCs) cultivated on silk scaffolds in response to different modes of electrostimulation (e.g., exogeneous and/or endogeneous). Endogeneous electrophysiology was altered through the use of monensin (10 nM) and glibenclamide (10 μM), along with external electrostimulation (60 kHz; 100-500 mV). Monensin enhanced the expression of early osteogenic markers such as alkaline phosphatase (ALP) and runt-related transcription factor 2 (RUNX-2). When exogeneous electrostimulation was combined with glibenclamide, more mature osteogenic marker upregulation based on bone sialoprotein expression (BSP) and mineralization was found. These results suggest the potential to exploit both exogeneous and endogeneous biophysical control of cell functions towards tissue-specific goals., (© 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Silk fibroin from the domesticated silkworm Bombyx mori is a naturally occurring biopolymer with charged hydrophilic terminal regions that end-cap a hydrophobic core consisting of repeating sequences of glycine, alanine, and serine residues. Taking inspiration from mussels that produce proteins rich in L-3,4-dihydroxyphenylalanine (DOPA) to adhere to a variety of organic and inorganic surfaces, the silk fibroin was functionalized with catechol groups. Silk fibroin was selected for its high molecular weight, tunable mechanical and degradation properties, aqueous processability, and wide availability. The synthesis of catechol-functionalized silk fibroin polymers containing varying amounts of hydrophilic polyethylene glycol (PEG, 5000 g/mol) side chains was carried out to balance silk hydrophobicity with PEG hydrophilicity. The efficiency of the catechol functionalization reaction did not vary with PEG conjugation over the range studied, although tuning the amount of PEG conjugated was essential for aqueous solubility. Adhesive bonding and cell compatibility of the resulting materials were investigated, where it was found that incorporating as little as 6 wt % PEG prior to catechol functionalization resulted in complete aqueous solubility of the catechol conjugates and increased adhesive strength compared with silk lacking catechol functionalization. Furthermore, PEG-silk fibroin conjugates maintained their ability to form β-sheet secondary structures, which can be exploited to reduce swelling. Human mesenchymal stem cells (hMSCs) proliferated on the silks, regardless of PEG and catechol conjugation. These materials represent a protein-based approach to catechol-based adhesives, which we envision may find applicability as biodegradable adhesives and sealants.

Bioelectric signaling is currently being explored as a novel regulator of cell processes in non-excitable cells. In particular, stem cells have demonstrated increasing evidence of electrophysiology-mediated regulation of stemness acquisition, proliferation, differentiation, and migration. However, in light of many reports of primary stem cell heterogeneity, it is important to characterize the variability of stem cell response to biophysical manipulations in order to assess the utility of bioelectric modulation as a universal strategy for stem cell control. In this work, human mesenchymal stem cells (hMSCs) from five donors were evaluated for their response to membrane potential (Vmem) depolarization. We compared the inter-donor variability of their osteogenic and adipogenic differentiation potential, as well as their ability to maintain a differentiated phenotype after induction. We identified the markers that responded most consistently across donors and found that calcium deposition and gene expression of bone sialoprotein, lipoprotein lipase, and fatty acid binding protein 4 are the preferred markers for assessing differentiation response to Vmem depolarization. We also note that since there exists variability even among some of these markers, these assays should be performed on any newly acquired hMSC population if their bioelectric properties are to be studied further.

A current focus of tissue engineering is the use of adult human mesenchymal stem cells (hMSCs) as an alternative to autologous chondrocytes for cartilage repair. Several natural and synthetic polymers (including cellulose) have been explored as a biomaterial scaffold for cartilage tissue engineering. While bacterial cellulose (BC) has been used in tissue engineering, its lack of degradability in vivo and high crystallinity restricts widespread applications in the field. Recently we reported the formation of a novel bacterial cellulose that is lysozyme-susceptible and -degradable in vivo from metabolically engineered Gluconacetobacter xylinus. Here we report the use of this modified bacterial cellulose (MBC) for cartilage tissue engineering using hMSCs. MBC's glucosaminoglycan-like chemistry, combined with in vivo degradability, suggested opportunities to exploit this novel polymer in cartilage tissue engineering. We have observed that, like BC, MBC scaffolds support cell attachment and proliferation. Chondrogenesis of hMSCs in the MBC scaffolds was demonstrated by real-time RT-PCR analysis for cartilage-specific extracellular matrix (ECM) markers (collagen type II, aggrecan and SOX9) as well as histological and immunohistochemical evaluations of cartilage-specific ECM markers. Further, the attachment, proliferation, and differentiation of hMSCs in MBC showed unique characteristics. For example, after 4 weeks of cultivation, the spatial cell arrangement and collagen type-II and ACAN distribution resembled those in native articular cartilage tissue, suggesting promise for these novel in vivo degradable scaffolds for chondrogenesis., (Copyright © 2013 John Wiley & Sons, Ltd.)

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., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Non-viral gene delivery systems are important transport vehicles that can be safe and effective alternatives to currently available viral systems. A new family of multifunctional spider silk-based gene carriers was bioengineered and found capable of targeting human mesenchymal stem cells (hMSCs). These carriers successfully delivered DNA to the nucleus of these mammalian cells. The presence of specific functional sequences in the recombinant proteins, such as a nuclear localization sequence (NLS) of the large tumor (T) antigen of the Simian virus 40 (SV40 ), an hMSC high affinity binding peptide (HAB), and a translocation motif (TLM) of the hepatitis-B virus surface protein (PreS2), and their roles in mitigation and enhancement of gene transfection efficiency towards hMSCs were characterized. The results demonstrate that these bioengineered spider silk proteins serve as effective carriers, without the well-known complications associated with viral delivery systems., (© 2014 Wiley Periodicals, Inc.)

Corneal tissue displays the highest peripheral nerve density in the human body. Engineering of biomaterials to promote interactions between neurons and corneal tissue could provide tissue models for nerve/cornea development, platforms for drug screening, as well as innovative opportunities to regenerate cornea tissue. The focus of this study was to develop a coculture system for differentiated human corneal stromal stem cells (dhCSSCs) and dorsal root ganglion neurons (DRG) to mimic the human cornea tissue interactions. Axon extension, connectivity, and neuron cell viability were studied. DRG neurons developed longer axons when cocultured with dhCSSCs in comparison to neuron cultures alone. To assess the mechanism involved in the coculture response, nerve growth factors (NGF) secreted by dhCSSCs including NGF, brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and neurotrophin-3 were characterized with greater focus on BDNF secretion. DhCSSCs also secreted collagen type I, an extracellular matrix molecule favorable for neuronal outgrowth. This coculture system provides a slowly degrading silk matrix to study neuronal responses in concert with hCSSCs related to innervation of corneal tissue with utility toward human corneal nerve regeneration and associated diseases., (© 2015 Wiley Periodicals, Inc.)

Biomaterial substrates composed of semi-aligned electrospun fibers are attractive supports for the regeneration of connective tissues because the fibers are durable under cyclic tensile loads and can guide cell adhesion, orientation, and gene expression. Previous studies on supported electrospun substrates have shown that both fiber diameter and mechanical deformation can independently influence cell morphology and gene expression. However, no studies have examined the effect of mechanical deformation and fiber diameter on unsupported meshes. Semi-aligned large (1.75 μm) and small (0.60 μm) diameter fiber meshes were prepared from degradable elastomeric poly(esterurethane urea) (PEUUR) meshes and characterized by tensile testing and scanning electron microscopy (SEM). Next, unsupported meshes were aligned between custom grips (with the stretch axis oriented parallel to axis of fiber alignment), seeded with C3H10T1/2 cells, and subjected to a static load (50 mN, adjusted daily), a cyclic load (4% strain at 0.25 Hz for 30 min, followed by a static tensile loading of 50 mN, daily), or no load. After 3 days of mechanical stimulation, confocal imaging was used to characterize cell shape, while measurements of deoxyribonucleic acid (DNA) content and messenger ribonucleic acid (mRNA) expression were used to characterize cell retention on unsupported meshes and expression of the connective tissue phenotype. Mechanical testing confirmed that these materials deform elastically to at least 10%. Cells adhered to unsupported meshes under all conditions and aligned with the direction of fiber orientation. Application of static and cyclic loads increased cell alignment. Cell density and mRNA expression of connective tissue proteins were not statistically different between experimental groups. However, on large diameter fiber meshes, static loading slightly elevated tenomodulin expression relative to the no load group, and tenascin-C and tenomodulin expression relative to the cyclic load group. These results demonstrate the feasibility of maintaining cell adhesion and alignment on semi-aligned fibrous elastomeric substrates under different mechanical conditions. The study confirms that cell morphology is sensitive to the mechanical environment and suggests that expression of select connective tissue genes may be enhanced on large diameter fiber meshes under static tensile loads.

There is significant clinical demand for functional tendon grafts in human and veterinary medicine. Tissue engineering techniques combining cells, scaffolds, and environmental stimuli may circumvent the shortcomings of traditional transplantation processes. In this study, the influence of cyclic mechanical stimulation on graft maturation and cellular phenotype was assessed in an equine model. Decellularized tendon scaffolds from four equine sources were seeded with syngeneic bone marrow-derived mesenchymal stem cells and subjected to 0%, 3%, or 5% strain at 0.33 Hz for up to 1 h daily for 11 days. Cells cultured at 3% strain integrated deep into their scaffolds, altered extracellular matrix composition, adopted tendon-like gene expression profiles, and increased construct elastic modulus and ultimate tensile strength to native levels. This bioreactor protocol is therefore suitable for cultivating replacement tendon material or as an in vitro model for studying differentiation of stem cells toward tendon., (© 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Three-dimensional (3-D) porous silk scaffolds with good biocompatibility and minimal immunogenicity show promise in a range of tissue regeneration applications. However, the challenge remains to effectively fabricate their microstructures and mechanical properties to satisfy the specific requirements of different tissues. In this study, silk scaffolds were fabricated to form an extracellular matrix (ECM) mimetic nanofibrous architecture using a mild process. A slowly increasing concentration process was applied to regulate silk self-assembly into nanofibers in aqueous solution. Then glycerol was blended with the nanofiber solution and induced silk crystallization in the lyophilization process, endowing freeze-dried scaffolds with water stability. The glycerol was leached from the scaffolds, leaving a similar porous structure at the micrometer scale but different topographies at the nanoscale. Compared to previous salt-leached and methanol-annealed scaffolds, the present scaffolds showed lower β-sheet content, softer mechanical property and improved cell growth and differentiation behaviors, suggesting their promising future as platforms for controlling stem cell fate and soft tissue regeneration., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Curcumin, a natural phenolic compound derived from the plant Curcuma longa, was physically entrapped and stabilized in silk hydrogel films, and its influence on human bone marrow-derived mesenchymal stem cells (hBMSC) was assessed related to adipogenic differentiation. The presence of curcumin significantly reduced the silk gelation time and changed the porous morphology of gel matrix, but did not change the formation of the silk beta-sheet structure. Based on spectrofluorimetric analysis, curcumin most likely interacted with hydrophobic residues in silk, interacting with the beta-sheet domains formed in the hydrogels. The antioxidant activity of silk film-associated curcumin remained functional over at least one month in both the dry and hydrated state. Negligible curcumin was released from silk hydrogel films over 48 h incubation in aqueous solution. For hBMSC cultured on silk films containing more than 0.25 mg ml(-1) curcumin, cell proliferation was inhibited, while adipogenesis was significantly promoted based on transcripts as well as Oil Red O staining. When hBMSC were cultured in media containing free curcumin, both proliferation and adipogenesis of hBMSC were inhibited when curcumin concentrations exceeded 5 μM, which is more than 1000 times higher than the level of curcumin released from the films in aqueous solution. Thus, silk film-associated curcumin exhibited different effects on hBMSC proliferation and differentiation compared with curcumin in solution., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Object: Neurocan is a major form of growth-inhibitory molecule (growth-IM) that suppresses axonal regeneration after neural injury. Bone marrow stromal cells (MSCs) have been shown to inhibit neurocan expression in vitro and in animal models of cerebral ischemia. Therefore, the present study was designed to investigate the effects of treatment of MSCs impregnated with collagen scaffolds on neurocan expression after traumatic brain injury (TBI)., Methods: Adult male Wistar rats were injured with controlled cortical impact and treated with saline, human MSCs (hMSCs) (3 × 10(6)) alone, or hMSCs (3 × 10(6)) impregnated into collagen scaffolds (scaffold + hMSCs) transplanted into the lesion cavity 7 days after TBI (20 rats per group). Rats were sacrificed 14 days after TBI, and brain tissues were harvested for immunohistochemical studies, Western blot analyses, laser capture microdissections, and quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) to evaluate neurocan protein and gene expressions after various treatments., Results: Animals treated with scaffold + hMSCs after TBI showed increased axonal and synaptic densities compared with the other groups. Scaffold + hMSC treatment was associated with reduced TBI-induced neurocan protein expression and upregulated growth-associated protein 43 (GAP-43) and synaptophysin expression in the lesion boundary zone. In addition, animals in the scaffold + hMSC group had decreased neurocan transcription in reactive astrocytes after TBI. Reduction of neurocan expression was significantly greater in the scaffold + hMSC group than in the group treated with hMSCs alone., Conclusions: The results of this study show that transplanting hMSCs with scaffolds enhances the effect of hMSCs on axonal plasticity in TBI rats. This enhanced axonal plasticity may partially be attributed to the downregulation of neurocan expression by hMSC treatment after injury.

How do we quantify cellular alignment? Cellular alignment is an important technique used to study and promote tissue regeneration in vitro and in vivo. Indeed, regenerative outcomes are often strongly correlated with the efficacy of alignment, making quantitative, automated assessment an important goal for the field of tissue engineering. There currently exist various classes of algorithms, which effectively address the problem of quantifying individual cellular alignments using Fourier methods, kernel methods, and elliptical approximation; however, these algorithms often yield population distributions and are limited by their inability to yield a scalar metric quantifying the efficacy of alignment. The current work builds on these classes of algorithms by adapting the signal processing methods previously used by our group to study the alignment of cellular processes. We use an automated, ellipse-fitting algorithm to approximate cell body alignment with respect to a silk biomaterial scaffold, followed by the application of the normalized cumulative periodogram criterion to produce a scalar value quantifying alignment. The proposed work offers a generalized method for assessing cellular alignment in complex, two-dimensional environments. This method may also offer a novel alternative for assessing the alignment of cell types with polarity, such as fibroblasts, endothelial cells, and mesenchymal stem cells, as well as nuclei., (© 2013 Wiley Periodicals, Inc.)

Although adult stem cell transplantation has been implemented as a therapy for tissue repair, it is limited by the availability of functional adult stem cells. A potential approach to generate stem and progenitor cells may be to modulate the differentiated status of somatic cells. Therefore, there is a need for a better understanding of how the differentiated phenotype of mature cells is regulated. We hypothesize that bioelectric signaling plays an important role in the maintenance of the differentiated state, as it is a functional regulator of the differentiation process in various cells and tissues. In this study, we asked whether the mature phenotype of osteoblasts and adipocytes derived from human mesenchymal stem cells (hMSCs) could be altered by modulation of their membrane potential. hMSC-derived osteoblasts and adipocytes were depolarized by treatment with ouabain, a Na(+)/K(+) ATPase inhibitor, or by treatment with high concentrations of extracellular K(+). To characterize the effect of voltage modulation on the differentiated state, the depolarized cells were evaluated for (1) the loss of differentiation markers; (2) the up-regulation of stemness markers and stem properties; and (3) differences in gene expression profiles in response to voltage modulation. hMSC-derived osteoblasts and adipocytes exhibited significant down-regulation of bone and fat tissue markers in response to depolarization, despite the presence of differentiation-inducing soluble factors, suggesting that bioelectric signaling overrides biochemical signaling in the maintenance of cell state. Suppression of the osteoblast or adipocyte phenotype was not accompanied by up-regulation of genes associated with the stem state. Thus, depolarization does not activate the stem cell genetic signature and, therefore, does not induce a full reprogramming event. However, after transdifferentiating the depolarized cells to evaluate for multi-lineage potential, depolarized osteoblasts demonstrated improved ability to achieve correct adipocyte morphology compared with nondepolarized osteoblasts. The present study thus demonstrates that depolarization reduces the differentiated phenotype of hMSC-derived cells and improves their transdifferentiation capacity, but does not restore a stem-like genetic profile. Through global transcript profiling of depolarized osteoblasts, we identified pathways that may mediate the effects of voltage signaling on cell state, which will require a detailed mechanistic inquiry in future studies.

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.

3D-biomaterial scaffolds with aligned architecture are of vital importance in tissue regeneration. A generic method is demonstrated to produce aligned biomaterial scaffolds using the physics of directional ice freezing. Homogeneously aligned 3D silk scaffolds with high porosity and alignment are prepared. The method can be adapted to a wide range of polymers and is devoid of any chemical reactions, thus avoiding potential complications associated with by-products. Mechanical properties and cellular responses with chondrocytes and bone-marrow-derived hMSCs are studied, assessing survival, proliferation, and differentiation. In vivo tests suggest biocompatibility of the matrices for future tissue engineering applications, specifically in areas where high cellular alignment is needed., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Collagen-like proteins in the bacteria Streptococcus pyogenes adopt a triple-helix structure with a thermal stability similar to that of animal collagens, can be expressed in high yield in Escherichia coli and can be easily modified through molecular biology techniques. However, potential applications for such recombinant collagens are limited by their lack of higher order structure to achieve the physical properties needed for most biomaterials. To overcome this problem, the S. pyogenes collagen domain was fused to a repetitive Bombyx mori silk consensus sequence, as a strategy to direct specific non-covalent binding onto solid silk materials whose superior stability, mechanical and material properties have been previously established. This approach resulted in the successful binding of these new collagen-silk chimeric proteins to silk films and porous scaffolds, and the binding affinity could be controlled by varying the number of repeats in the silk sequence. To explore the potential of collagen-silk chimera for regulating biological activity, integrin (Int) and fibronectin (Fn) binding sequences from mammalian collagens were introduced into the bacterial collagen domain. The attachment of bioactive collagen-silk chimeras to solid silk biomaterials promoted hMSC spreading and proliferation substantially in comparison to the controls. The ability to combine the biomaterial features of silk with the biological activities of collagen allowed more rapid cell interactions with silk-based biomaterials, improved regulation of stem cell growth and differentiation, as well as the formation of artificial extracellular matrices useful for tissue engineering applications., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The alignment and osteogenic differentiation of MSCs on patterned silk films (PF) is investigated as a bottom-up approach toward engineering bone lamellae. Screening PF with various groove dimensions shows that cell alignment is mediated by both the pattern width and depth. MSCs are differentiated in osteogenic medium for four weeks on flat films and on the PF that produce the best alignment. The PF support osteogenic differentiation while also inducing lamellar alignment of cells and matrix deposition. A secondary alignment effect is noted on the PF where a new layer of aligned cells grows over the first layer, but rotated obliquely to the underlying pattern. This layering and rotation of the MSCs resembles the cellular organization observed in native lamellar bone., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

3D silk/HA microperiodic scaffolds for bone tissue engineering and angiogenesis are fabricated by direct-write assembly. This approach can be used to control filament and spacing size in the scaffold to allow investigation of the effect of scaffold architecture on osteogenesis and vessel-like structure formation from stem cells and endothelial cells., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The response of human bone marrow derived human mesenchymal stem cells (hMSCs) encapsulated in silk ionomer hydrogels was studied. Silk aqueous solutions with silk-poly-L-lysine or silk-poly-L-glutamate were formed into hydrogels via ultrasonication in situ with different net charges. hMSCs were encapsulated within the hydrogels and the impact of matrix charge was assessed over weeks in osteogenic, adipogenic and maintenance growth media. These modified silk charged polymers supported cell viability and proliferative potential, and the hMSCs were able to differentiate toward osteogenic or adipogenic lineages in the corresponding differentiation media. The silk/silk-poly-L-lysine hydrogels exhibited a positive effect on selective osteogenesis of hMSCs, inducing differentiation toward an osteogenic lineage even in the absence of osteogenic supplements, while also inhibiting adipogenesis. In contrast, silk/silk fibroin-poly-L-glutamate hydrogels supported both osteogenic and adipogenic differentiation of hMSCs when cultured under induction conditions. The results demonstrate the potential utility of silk-based ionomers in gel formats for hMSCs encapsulation and for directing hMSCs long term functional differentiation toward specific lineages., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Non-invasive approaches to assess tissue function could improve significantly current methods to diagnose diseases and optimize engineered tissues. In this study, we describe a two-photon excited fluorescence microscopy approach that relies entirely on endogenous fluorophores to dynamically quantify functional metabolic readouts from individual cells within three-dimensional engineered tissues undergoing adipogenic differentiation over six months. Specifically, we employ an automated approach to analyze 3D image volumes and extract a redox ratio of metabolic cofactors. We identify a decrease in redox ratio over the first two months of culture that is associated with stem cell differentiation and lipogenesis. In addition, we demonstrate that the presence of endothelial cells facilitate greater cell numbers deeper within the engineered tissues. Since traditional assessments of engineered tissue structure and function are destructive and logistically intensive, this non-destructive, label-free approach offers a potentially powerful high-content characterization tool for optimizing tissue engineering protocols and assessing engineered tissue implants., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Adipose tissue-derived stem cells (ASCs) are considered as an attractive stem cell source for tissue engineering and regenerative medicine. We compared human bone marrow-derived mesenchymal stem cells (hMSCs) and hASCs under dynamic hydraulic compression to evaluate and compare osteogenic abilities. A novel micro cell chip integrated with microvalves and microscale cell culture chambers separated from an air-pressure chamber was developed using microfabrication technology. The microscale chip enables the culture of two types of stem cells concurrently, where each is loaded into cell culture chambers and dynamic compressive stimulation is applied to the cells uniformly. Dynamic hydraulic compression (1 Hz, 1 psi) increased the production of osteogenic matrix components (bone sialoprotein, oateopontin, type I collagen) and integrin (CD11b and CD31) expression from both stem cell sources. Alkaline phosphatase and Alrizarin red staining were evident in the stimulated hMSCs, while the stimulated hASCs did not show significant increases in staining under the same stimulation conditions. Upon application of mechanical stimulus to the two types of stem cells, integrin (β1) and osteogenic gene markers were upregulated from both cell types. In conclusion, stimulated hMSCs and hASCs showed increased osteogenic gene expression compared to non-stimulated groups. The hMSCs were more sensitive to mechanical stimulation and more effective towards osteogenic differentiation than the hASCs under these modes of mechanical stimulation.

Despite the decline in U.S. cancer incidence and mortality rates, cancer remains the number one cause of death for people under the age of 85 and one in four people in the U.S. will die of cancer, mainly because of metastasis. Recently, interest in mesenchymal stem cell (MSC) tumor-homing has led to inquires into: (a) why MSCs home to tumors, (b) what the inherent protumor and antitumor consequences are, and (c) how to best capitalize on MSC tumor-homing for cell-based diagnostics and therapy. Here, these questions are reviewed and method for addressing them using animal models and tracking methodologies (or, synonymously, detection methodologies) are discussed. First, MSCs in a regenerative and tumor-homing context are reviewed, followed by MSC delivery and genetic labeling methods for tissue model systems. Finally, the use of the nonoptical methods, magnetic resonance imaging, positron emission tomography, and single photon emission computed tomography, along with optical methods, fluorescence imaging and bioluminescent imaging, are reviewed related to tracking MSCs within disease model settings. The benefits and drawbacks of each detection method in animal models is reviewed along with the utility of each for therapeutic use., (Copyright © 2011 AlphaMed Press.)

A new class of biomimetic, bioresorbable apatitic calcium phosphate cement (CPC) was recently developed. The handling characteristics, and the ability to harden at body temperature in the presence of physiological saline, make this material an attractive clinical bone substitute and delivery vehicle for therapeutic agents in orthopedic applications. The major challenge with the material is formulating an injectable paste with options for cell delivery, in order to regenerate new bone faster and with high quality. In this study, three different additives and/or viscosity modifiers (carboxymethylcellulose, silk, and alginate) were incorporated into a CPC matrix. Injectability, cell viability, cell proliferation, surface morphology, and gene expression for osteogenesis of hMSCs were all evaluated. Injectable CPC-gel composites with cell protection were achieved. The CPC modified with alginate provided the best results based on cell proliferation, ALP and collagen production, and osteogenic transcript increases (for ALP, type I collagen, BSP, and OP). Furthermore, osteogenic analysis indicated lineage-specific differentiation of hMSCs into osteogenic outcomes. The results suggest that CPC mixed with alginate can be used as a cell delivery vehicle for bone regeneration., (Copyright © 2010 Wiley Periodicals, Inc.)

Background: Electric fields are integral to many biological events, from maintaining cellular homeostasis to embryonic development to healing. The application of electric fields offers substantial therapeutic potential, while optimal dosing regimens and the underlying mechanisms responsible for the positive clinical impact are poorly understood., Methods: The purpose of this study was to track the differentiation profile and stress response of human bone marrow derived mesenchymal stem cells (hMSCs) undergoing osteogenic differentiation during exposure to a 20 mV/cm, 60 kHz electric field. Morphological and biochemical changes were imaged using endogenous two-photon excited fluorescence (TPEF) and quantitatively assessed through eccentricity calculations and extraction of the redox ratio from NADH, FAD and lipofuscin contributions. Real time reverse transcriptase-polymerase chain reactions (RT-PCR) were used to track osteogenic differentiation markers, namely alkaline phosphatase (ALP) and collagen type 1 (col1), and stress response markers, such as heat shock protein 27 (hsp27) and heat shock protein 70 (hsp70). Comparisons of collagen deposition between the stimulated hMSCs and controls were examined through second harmonic generation (SHG) imaging., Results: Quantitative differences in cell morphology, as described through an eccentricity ratio, were found on days 2 and days 5 (p < 0.05) in samples exposed to the electric field. A delayed but two fold increase in ALP and col1 transcript was detected by week 2 (p < 0.05) in differentiating hMSCs exposed to an electric field in comparison to the nonstimulated controls. Upregulation in stress marker, hsp27, and type 1 collagen deposition were correlated with this response. Increases in NADH, FAD, and lipofuscin were traced in the stimulation group during the first week of field exposure with differences statistically significant on day 10 (p < 0.05). Changes in hsp27 expression correlate well with changes in lipofuscin detected in the stimulation group, suggesting a connection with oxidative stress. Both differentiation factors and electrical stimulation improved hMSC differentiation potential to bone based on calcium deposition on day 28., Conclusions: Electrical stimulation is a useful tool to improve hMSC osteogenic differentiation, while heat shock proteins may reveal underlying mechanisms, and optical non-invasive imaging may be used to monitor the induced morphological and biochemical changes.

Tissue engineered cartilage can be grown in vitro with the use of cell-scaffold constructs and bioreactors. The present study was designed to investigate the effects of perfusion bioreactors on the chondrogenic potential of engineered constructs prepared from porous silk fibroin scaffolds seeded with human embryonic stem cell (hESC)-derived mesencyhmal stem cells (MSCs). After four weeks of incubation, constructs cultured in perfusion bioreactors showed significantly higher amounts of glycosaminoglycans (GAGs) (p < 0.001), DNA (p < 0.001), total collagen (p < 0.01), and collagen II (p < 0.01) in comparison to static culture. Mechanical stiffness of constructs increased 3.7-fold under dynamic culture conditions and RT-PCR results concluded that cells cultured in perfusion bioreactors highly expressed (p < 0.001) cartilage-related genes when compared with static culture. Distinct differences were noted in tissue morphology, including polygonal extracellular matrix structure of engineered constructs in thin superficial zones and an inner zone under static and dynamic conditions, respectively. The results suggest that the utility of perfusion bioreactors to modulate the growth of tissue-engineered cartilage and enhance tissue growth in vitro., (Copyright © 2010 Wiley Periodicals, Inc.)

Silk fibroin protein is biodegradable and biocompatible, exhibiting excellent mechanical properties for various biomedical applications. However, porous three-dimensional (3-D) silk fibroin scaffolds, or silk sponges, usually fall short in matching the initial mechanical requirements for bone tissue engineering. In the present study, silk sponge matrices were reinforced with silk microparticles to generate protein-protein composite scaffolds with desirable mechanical properties for in vitro osteogenic tissue formation. It was found that increasing the silk microparticle loading led to a substantial increase in the scaffold compressive modulus from 0.3 MPa (non-reinforced) to 1.9 MPa for 1:2 (matrix:particle) reinforcement loading by dry mass. Biochemical, gene expression, and histological assays were employed to study the possible effects of increasing composite scaffold stiffness, due to microparticle reinforcement, on in vitro osteogenic differentiation of human mesenchymal stem cells (hMSCs). Increasing silk microparticle loading increased the osteogenic capability of hMSCs in the presence of bone morphogenic protein-2 (BMP-2) and other osteogenic factors in static culture for up to 6 weeks. The calcium adsorption increased dramatically with increasing loading, as observed from biochemical assays, histological staining, and microcomputer tomography (μCT) analysis. Specifically, calcium content in the scaffolds increased by 0.57, 0.71, and 1.27 mg (per μg of DNA) from 3 to 6 weeks for matrix to particle dry mass loading ratios of 1:0, 1:1, and 1:2, respectively. In addition, μCT imaging revealed that at 6 weeks, bone volume fraction increased from 0.78% for non-reinforced to 7.1% and 6.7% for 1:1 and 1:2 loading, respectively. Our results support the hypothesis that scaffold stiffness may strongly influence the 3-D in vitro differentiation capabilities of hMSCs, providing a means to improve osteogenic outcomes., (Copyright © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

American women have a nearly 25% lifetime risk of developing breast cancer, with 20% to 40% of these patients developing life-threatening metastases. More than 70% of patients presenting with metastases have skeletal involvement, which signals progression to an incurable stage. Tumor-stroma cell interactions are only superficially understood, specifically regarding the ability of stromal cells to affect metastasis. In vivo models show that exogenously supplied human bone marrow-derived stem cells (hBMSC) migrate to breast cancer tumors, but no reports have shown endogenous hBMSC migration from the bone to primary tumors. Here, we present a model of in vivo hBMSC migration from a physiologic human bone environment to human breast tumors. Furthermore, hBMSCs alter tumor growth and bone metastasis frequency. These may home to certain breast tumors based on tumor-derived TGF-β1. Moreover, at the primary tumor level, interleukin 17B (IL-17B)/IL-17BR signaling may mediate interactions between hBMSCs and breast cancer cells., (©2010 AACR.)

Tailoring tissue engineering strategies to match patient- and tissue-specific bone regeneration needs offers to improve clinical outcomes. As a step toward this goal, osteogenic outcomes and metabolic parameters were assessed when varying inputs into the bone formation process. Silk protein scaffolds seeded with human mesenchymal stem cells in osteogenic differentiation media were used to study in vitro osteogenesis under varied conditions of amino acid (lysine and proline) concentration and oxygen level. The cells were assessed to probe how the microenvironment impacted metabolic pathways and thus osteogenesis. The most favorable osteogenesis outcomes were found in the presence of low (5%) oxygen combined with high lysine and proline concentrations during in vitro cultivation. This same set of culture conditions also showed the highest glucose consumption, lactate synthesis, and certain amino acid consumption rates. On the basis of these results and known pathways, a holistic metabolic model was derived which shows that lysine and proline supplements as well as low (5%) oxygen levels regulate collagen matrix synthesis and thereby rates of osteogenesis. This study establishes early steps toward a foundation for patient- and tissue-specific matches between metabolism, repair site, and tissue engineering approaches toward optimized bone regeneration.

Large-scale tissue engineering is limited by nutrient perfusion and mass transport limitations, especially oxygen diffusion, which restrict construct development to smaller than clinically relevant dimensions and limit the ability for in vivo integration. The goal of this work was to develop a modular approach to tissue engineering, where scaffold and tissue size, transport issues, and surgical implantation in vivo are considered from the outset. Human mesenchymal stem cells (hMSCs) were used as the model cell type, as their differentiation has been studied for several different cell lineages and often with conflicting results. Changes in the expression profiles of hMSCs differentiated under varied oxygen tensions are presented, demonstrating tissue-specific oxygen requirements for both adipogenic (20% O₂) and chondrogenic (5% O₂) differentiation. Oxygen and nutrient transport were enhanced by developing a bioreactor system for perfusing hMSC-seeded collagen gels using porous silk tubes, resulting in enhanced oxygen transport and cell viability within the gels. These systems are simple to use and scaled for versatility, to allow for the systematic study of relationships between cell content, oxygen, and cell function. The data may be combined with oxygen transport modeling to derive minimally sized modular units for construction of clinically relevant tissue-engineered constructs, a generic strategy that may be employed for vascularized target tissues.

Background: The engineering of functional tissues is a complex multi-stage process, the success of which depends on the careful control of culture conditions and ultimately tissue maturation. To enable the efficient optimization of tissue development protocols, techniques suitable for monitoring the effects of added stimuli and induced tissue changes are needed., Methodology/principal Findings: Here, we present the quantitative use of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) as a noninvasive means to monitor the differentiation of human mesenchymal stem cells (hMSCs) using entirely endogenous sources of contrast. We demonstrate that the individual fluorescence contribution from the intrinsic cellular fluorophores NAD(P)H, flavoproteins and lipofuscin can be extracted from TPEF images and monitored dynamically from the same cell population over time. Using the redox ratio, calculated from the contributions of NAD(P)H and flavoproteins, we identify distinct patterns in the evolution of the metabolic activity of hMSCs maintained in either propagation, osteogenic or adipogenic differentiation media. The differentiation of these cells is mirrored by changes in cell morphology apparent in high resolution TPEF images and by the detection of collagen production via SHG imaging. Finally, we find dramatic increases in lipofuscin levels in hMSCs maintained at 20% oxygen vs. those in 5% oxygen, establishing the use of this chromophore as a potential biomarker for oxidative stress., Conclusions/significance: In this study we demonstrate that it is possible to monitor the metabolic activity, morphology, ECM production and oxidative stress of hMSCs in a non-invasive manner. This is accomplished using generally available multiphoton microscopy equipment and simple data analysis techniques, such that the method can widely adopted by laboratories with a diversity of comparable equipment. This method therefore represents a powerful tool, which enables researchers to monitor engineered tissues and optimize culture conditions in a near real time manner.

The development of biomaterials that mimic the physiological binding of growth factors to the extracellular matrix (ECM) is an appealing strategy for advanced growth factor delivery systems. In vivo, fibroblast growth factor 2 (FGF-2) binds to the sulfated glycosaminoglycan heparan sulfate, which is a major component of the ECM. Therefore, we tested whether silk fibroin (SF) decorated with a sulfonated moiety could mimic the natural ECM environment and lead to advanced delivery of this heparin-binding growth factor. Using a diazonium coupling reaction, modified SF derivatives containing approximately 20, 40, 55 and 70 sulfonic acid groups per SF molecule were obtained. Films of the SF derivative decorated with 70 sulfonic acid groups per SF molecule resulted in a 2-fold increase in FGF-2 binding as compared to native SF. More than 99% of bound FGF-2 could be retained on all SF derivatives. However, protection of FGF-2 potency was only achieved with at least 40 sulfonic acid groups per SF molecule, as observed by reduced metabolic activity and enhanced levels of phosphorylated extracellular signal-regulated kinases (pERK1/2) in cultured human mesenchymal stem cells (hMSCs). This study introduces a first step towards the development of an ECM-mimicking biomaterial for sustained, non-covalent binding, controlled delivery and preserved potency of biomolecules., ((c) 2009 Elsevier Ltd. All rights reserved.)

Mesenchymal condensation is a pre-requisite of chondrogenesis during embryonic development. The current understanding of chondrogenesis is limited in terms of chondrogenic condensation mechanisms. In particular, the role of matrix stiffness on homotypic cell-cell interactions leading to the establishment of distinct aggregated chondrogenic morphology from mesenchymal cells is unclear. An in vitro biomaterials-based model to assess the interactions of matrix stiffness on chondrogensis is described herein, where by sensing subtle variation in morphology and stiffness of nanofibrous silk protein matrixes human mesenchymal stem cells migrated and assumed aggregated morphologies, mimicking early stage chondrogenesis. This simple in vitro model system has potential to play a significant role to gain insight into underlying mechanisms of mesenchymal condensation steps during chondrogenesis, integrating concepts of developmental biology, biomaterials and tissue engineering.

Cartilage tissue can be engineered by starting from a diversity of cell sources, including stem-cell based and primary cell-based platforms. Selecting an appropriate cell source for the process of cartilage tissue engineering or repair is critical and challenging, due to the variety of cell options available. In this study, cellular responses of isolated human chondrocytes, human embryonic stem cells and mesenchymal stem cells (MSCs) derived from three sources, human embryonic stem cells, bone marrow and adipose tissue, were assessed for chondrogenic potential in 3D culture. All cell sources were characterized by FACS analysis to compare expression of some surface markers. The cells were differentiated in two different biomaterial matrices, silk and chitosan scaffolds, in the presence and absence of bone morphogenetic protein 6 (BMP6), along with the standard chondrogenic differentiating factors. Embryonic stem cells-derived MSCs showed unique characteristics, with preserved chondrogenic phenotype in both scaffolds with regard to chondrogenesis, as determined by real time RT-PCR, histological and microscopical analyses. After 4 weeks of cultivation, embryonic stem cells-derived MSCs were promising for chondrogenesis, particularly in the silk scaffolds with BMP6. The results suggest that cell source differences are important to consider with regard to chondrogenic outcomes, and among the variables addressed here the human embryonic stem cells-derived MSCs were the preferred cell source.

A novel generation of silk-based brain implants engineered to release adenosine was recently shown to provide robust seizure suppression in kindled rats. As a first step to develop stem cell-coated silk-based 3D-scaffolds for the therapeutic long-term delivery of adenosine we engineered human mesenchymal stem cells (hMSCs) to release adenosine. Here we demonstrate reduction of chronic seizures in a mouse model of CA3-selective epileptogenesis after infrahippocampal transplantation of adenosine-releasing hMSCs.

Human mesenchymal stem cells (hMSCs) isolated from bone marrow aspirates were cultured on silk scaffolds in rotating bioreactors for three weeks with either chondrogenic or osteogenic medium supplements to engineer cartilage- or bone-like tissue constructs. Osteochondral composites formed from these cartilage and bone constructs were cultured for an additional three weeks in culture medium that was supplemented with chondrogenic factors, supplemented with osteogenic factors or unsupplemented. Progression of cartilage and bone formation and the integration between the two regions were assessed by medical imaging (magnetic resonance imaging and micro-computerized tomography imaging), and by biochemical, histological and mechanical assays. During composite culture (three to six weeks), bone-like tissue formation progressed in all three media to a markedly larger extent than cartilage-like tissue formation. The integration of the constructs was most enhanced in composites cultured in chondrogenic medium. The results suggest that tissue composites with well-mineralized regions and substantially less developed cartilage regions can be generated in vitro by culturing hMSCs on silk scaffolds in bioreactors, that hMSCs have markedly higher capacity for producing engineered bone than engineered cartilage, and that chondrogenic factors play major roles at early stages of bone formation by hMSCs and in the integration of the two tissue constructs into a tissue composite.

A simple chemical modification method using diazonium coupling chemistry was developed to tailor the structure and hydrophilicity of silk fibroin protein. The extent of modification using several aniline derivatives was characterized using UV-vis and 1H NMR spectroscopies, and the resulting protein structure was analyzed with ATR-FTIR spectroscopy. Introduction of hydrophobic functional groups facilitated rapid conversion of the protein from a random coil to a beta-sheet structure, while addition of hydrophilic groups inhibited this process. hMSCs were grown on these modified silks to assess the biocompatibility of these materials. The hydrophilicity of the silk derivatives was found to affect the growth rate and morphology, but hMSCs were able to attach, proliferate and differentiate into an osteogenic lineage on all of the silk derivatives.