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In tissue engineering, the development of an appropriate scaffold is crucial to provide a framework for new tissue growth. The use of cryogels as scaffolds shows promise due to their macroporous structure, but the pore size, distribution, and interconnectivity is highly variable depending on the fabrication process. The objective of the current research is to provide a technique for controlled anisotropy in chitosan-gelatin cryogels to develop scaffolds for bone tissue engineering application. A mold was developed using additive manufacturing to be used during the freezing process in order to fabricate cryogels with a more interconnected pore structure. The scaffolds were tested to evaluate their porosity, mechanical strength, and to observe cell infiltration through the cryogel. It was found that the use of the mold allowed for the creation of designated pores within the cryogel structure which facilitated cell infiltration to the center of the scaffold without sacrificing mechanical integrity of the structure.
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Quitosana , Engenharia Tecidual , Engenharia Tecidual/métodos , Criogéis/química , Alicerces Teciduais/química , Quitosana/química , Gelatina/química , Anisotropia , PorosidadeRESUMO
Tissue-engineered scaffolds have been identified as appropriate templates for bone regeneration, especially complex geometries seen in craniofacial defects. Here we describe the general fabrication and modification of hydrogels, cryogels, and electrospun scaffolds. These scaffolds offer a variety of templates for facilitating bone growth and regeneration in craniofacial applications.
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Regeneração Óssea , Engenharia Tecidual , Osso e Ossos , Criogéis , Alicerces TeciduaisRESUMO
Microcomputed tomography is an important technique for distinguishing the vascular network from tissues with similar X-ray attenuation. Here, we describe a composite of barium sulfate (BaSO4) nanoparticles, calcium carbonate (CaCO3) nanoparticles, and alginate that provides improved performance over microscale BaSO4 particles, which are currently used clinically as X-ray contrast agents. BaSO4 and CaCO3 nanoparticles were synthesized using a polyol method with tetraethylene glycol as solvent and capping agent. The nanoparticles show good colloidal stability in aqueous solutions. A deliverable nanocomposite gel contrast agent was produced by encapsulation of the BaSO4 and CaCO3 nanoparticles in an alginate gel matrix. The gelation time was controlled by addition of d-(+)-gluconic acid δ-lactone, which controls the rate of dissolution of the CaCO3 nanoparticles that produce Ca2+ which cross-links the gel. Rapid cross-linking of the gel by Ba2+ was minimized by producing BaSO4 nanoparticles with an excess of surface sulfate. The resulting BaSO4-CaCO3 nanoparticle alginate gel mechanical properties were characterized, including the gel storage modulus, peak stress and elastic modulus, and radiodensity. The resulting nanocomposite has good viscosity control and good final gel stiffness. The nanocomposite has gelation times between 30 and 35 min, adequate for full body perfusion. This is the first nanoscale composite of a radiopaque metal salt to be developed in combination with an alginate hydrogel and designed for medical perfusion and vascular imaging applications.
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BACKGROUND: Platelet-rich-plasma (PRP) is used to treat knee osteoarthritis; however, mechanistic evidence of PRP effectiveness for pain relief is limited. OBJECTIVE: To assess molecular biomarkers and mesenchymal stem cells (MSCs) in synovial fluid during PRP treatment of the osteoarthritic knee joint. DESIGN: Single blinded, randomized, placebo controlled pilot study. SETTING: Veterans Affairs Medical Center. PARTICIPANTS: Seventeen participants with mild to moderate knee osteoarthritis were randomized in a 2:1 placebo-controlled ratio, receiving PRP or saline (placebo) intra-articular injection into the knee joint. METHODS: Knee synovial fluid was analyzed before the respective injections and again 10 days following injection. Participants were followed up to 12 months completing visual analog scale (VAS) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) questionnaires at intervals over that period. MAIN OUTCOME MEASURES: The effects of PRP on synovial protein and MSC gene expression levels were measured by multiplex enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. RESULTS: Novel biomarkers including levels of interleukin (IL)-5, IL-6, IL-10, and tumor necrosis factor-α were measured in synovial fluid 10 days after PRP treatment. Altered gene expression profiles in MSCs from patients treated with PRP were observed for matrix metalloproteinases and inflammatory markers (IL-6, IL-8, CCL2, TNF-α). A2M protease was significantly increased following PRP treatment (P = .005). WOMAC scores declined for up to 3 months from baseline levels and remained low at 6 and 12 months in the PRP group. In contrast, WOMAC scores for patients receiving the saline injection were relatively unchanged for up to 12 months. CONCLUSIONS: We report significant changes for the biomarker A2M (P = .005) as well as differences in expression of cellular markers and postulate that PRP modulates the local knee synovial environment by altering the inflammatory milieu, matrix degradation, and angiogenic growth factors. The PRP treatment group had less pain and stiffness and improved function scores.
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Osteoartrite do Joelho , Plasma Rico em Plaquetas , Humanos , Ácido Hialurônico , Injeções Intra-Articulares , Articulação do Joelho , Osteoartrite do Joelho/tratamento farmacológico , Resultado do TratamentoRESUMO
BACKGROUND: In transposition flaps, thicker tissue and higher degrees of rotation are associated with increased pivotal restraint; however, limited experimental data exist quantifying the degree to which these affect flap biomechanics. The use of artificial skin models in conjunction with digital image correlation technology allows for investigation into biomechanical properties of skin flaps. OBJECTIVE: To quantify the effects of tissue thickness and rotational angles on pivotal restraint within transposition flaps using artificial skin models. METHODS: Ninety degree bilobed and trilobed flaps were used to close defects in artificial skin models of increasing thicknesses. Digital image correlation was used to quantify strain. Quantitative and qualitative differences in strain were assessed in increasing flap thicknesses and between flap designs. RESULTS: Increasing flap thickness was associated with decreasing strain. In the bilobed flap, increasing thickness was associated with displacement of the flap pivot point away from the distal flap edge. Comparatively, lower angles of rotation in the trilobed flap were not associated with migration of the flap pivot point. CONCLUSION: Increased pivotal restraint observed in higher degrees of rotation is due to migration of the flap pivot point. This model supports the common practice of decreasing flap angles to compensate for pivotal restraint.
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Pele Artificial , Retalhos Cirúrgicos , Fenômenos Biomecânicos , Teste de Materiais , Fotografação , Rotação , Resistência à TraçãoRESUMO
Platelet-rich plasma (PRP) is an autologous blood product that contains a variety of growth factors (GFs) that are released upon platelet activation. Despite some therapeutic potential of PRP in vitro, in vivo data are not convincing. Bolus injection of PRP is cleared rapidly from the body diminishing its therapeutic efficacy. This highlights a need for a delivery vehicle for a sustained release of PRP to improve its therapeutic effect. In this study, we used microfluidics to fabricate biodegradable PRP-loaded polyethylene glycol (PEG) microspheres. PRP was incorporated into the microspheres as a lyophilized PRP powder either as is (powder PRP) or first solubilized and pre-clotted to remove clots (liquid PRP). A high PRP loading of 10% w/v was achieved for both PRP preparations. We characterized the properties of the resulting PRP-loaded PEG microspheres including swelling, modulus, degradation, and protein release as a function of PRP loading and preparation. Overall, loading powder PRP into the PEG microspheres significantly affected the properties of microspheres, with the most pronounced effect noted in degradation. We further determined that microsphere degradation in the presence of powder PRP was affected by platelet aggregation and clotting. Platelet aggregation did not prevent but prolonged sustained PRP release from the microspheres. The delivery system developed and characterized herein could be useful for the loading and releasing of PRP to promote tissue regeneration and wound healing or to suppress tissue degeneration in osteoarthritis, and intervertebral disc degeneration.
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Hydrogels that can respond to multiple external stimuli represent the next generation of advanced functional biomaterials. Here, a series of multimodal hydrogels were synthesized that can contract and expand reversibly over several cycles while changing their mechanical properties in response to blue and red light, as well as heat (â¼50 °C). The light-responsive behavior was achieved through a photoredox-based mechanism consisting of photoinduced electron transfer from a zinc porphyrin photocatalyst in its excited state to oligoviologen-based macrocrosslinkers, both of which were integrated into the hydrogel polymer network during gel formation. Orthogonal thermoresponsive properties were also realized by introducing N-isopropyl acrylamide (NIPAM) monomer simultaneously with hydroxyethyl acrylate (HEA) in the pre-gel mixture to produce a statistical 60 : 40 HEA : NIPAM polymer network. The resultant hydrogel actuators - crosslinked with either a styrenated viologen dimer (2V4+-St) or hexamer (6V12+-St) - were exposed to red or blue light, or heat, for up to 5 h, and their rate of contraction, as well as the corresponding changes in their physical properties (i.e., stiffness, tensile strength, Young's modulus, etc.), were measured. The combined application of blue light and heat to the 6V12+-St-based hydrogels was also demonstrated, resulting in hydrogels with more than two-fold faster contraction kinetics and dramatically enhanced mechanical robustness when fully contracted. We envision that the reported materials and the corresponding methods of remotely manipulating the dynamic hydrogels may serve as a useful blueprint for future adaptive materials used in biomedical applications.
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Osteons are the repeating unit throughout cortical bone, consisting of canals filled with blood and nerve vessels surrounded by concentric lamella of hydroxyapatite-containing collagen fibers, providing mechanical strength. Creating a biodegradable scaffold that mimics the osteon structure is crucial for optimizing cellular infiltration and ultimately the replacement of the scaffold with native cortical bone. In this study, a modified air-gap electrospinning setup was exploited to continuously wrap highly aligned polycaprolactone polymer nanofibers around individual 1393 bioactive glass microfibers, resulting in a synthetic structure similar to osteons. By varying the parameters of the device, scaffolds with polymer fibers wrapped at angles between 5-20° to the glass fiber were chosen. The scaffold indicated increased cell migration by demonstrating unidirectional cell orientation along the fibers, similar to recent work regarding aligned nerve and muscle regeneration. The wrapping decreased the porosity from 90% to 80%, which was sufficient for glass conversion through ion exchange validated by inductively coupled plasma. Scaffold degradation was not cytotoxic. Encapsulating the glass with polymer nanofibers caused viscoelastic deformation during three-point bending, preventing typical brittle glass fracture, while maintaining cell migration. This scaffold design structurally mimics the osteon, with the intent to replace its material compositions for better regeneration.
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Alginate is a highly tailorable, biocompatible polymer whose properties can be tuned to mimic the properties of native nucleus pulposus (NP) tissue. Platelet-rich plasma (PRP) is a highly accessible, inexpensive, and readily available mix of pro-regenerative factors. By functionalizing alginate with PRP, a mechanically optimized, bioactive alginate NP analogue may stimulate NP cells to proliferate and accumulate matrix over a longer period of time than if the PRP were solely encapsulated within the hydrogel. In this study, PRP was chemically bound to alginate using carbodiimide chemistry and mechanically, physically, and cytologically compared to plain alginate as well as alginate containing free-floating lyophilized PRP. The alginates were mechanically and physically characterized; PRP-conjugated alginate had similar mechanical properties to controls and had the benefit of retained PRP proteins within the hydrogel. Human nucleus pulposus cells (hNPCs) were seeded within the modified alginates and cultured for 14 days. Quantification data of glycosaminoglycans suggests that PRP-incorporated alginate has the potential to increase ECM production within the characterized alginate constructs, and that PRP-functionalized alginate can retain protein within the hydrogel over time. This is the first study to functionalize the milieu of PRP proteins onto alginate and characterize the mechanical and physical properties of the modified alginates. This study also incorporates hNPCs into the characterized PRP-modified alginates to observe phenotypic maintenance when encapsulated within the in situ gelling constructs.
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Alginatos/química , Materiais Biocompatíveis/química , Carbodi-Imidas/química , Hidrogéis/química , Plasma Rico em Plaquetas/química , Alicerces Teciduais/química , Proliferação de Células , Células Cultivadas , Matriz Extracelular/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Hidrogéis/uso terapêutico , Injeções , Fenômenos Mecânicos , Núcleo Pulposo/citologia , Engenharia TecidualRESUMO
Significance: Historically, honey has been regarded as a potent agent in bacterial inhibition and wound healing. An increased prevalence of antibiotic resistant pathogens spurred an initial resurgence in honey's clinical popularity, with it quickly finding a place in wound care and regenerative medicine. However, this renewed usage demanded a need for improved delivery and overall research of its bioactive properties. This review provides an overview of the antibacterial properties and clinical use of honey. Recent Advances: The past and present clinical use of honey is noted, focusing specifically on burns and ulcers, as these are the most common applications of the natural agent. While honey is often used without modification clinically, there are also commercially available products ranging from dressings to gels, which are discussed. Critical Issues: Despite these products growing in popularity, the need for improved delivery and a structure to support wound healing could improve the treatment method. Future Directions: Tissue engineering scaffolds can provide an alternative method of honey delivery with research focusing primarily on electrospun scaffolds, hydrogels, and cryogels. Current studies on these scaffolds are discussed with respect to their advantages and potential for future clinical work. Overall, this review provides a comprehensive overview of the properties of honey, its current use in wound healing, and the potential for future incorporation into tissue-engineered scaffolds to provide an innovative wound healing agent.
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There is a growing interest in being able to control the mechanical properties of hydrogels for applications in materials, medicine, and biology. Primarily, changes in the hydrogel's physical properties, i.e., stiffness, toughness, etc., are achieved by modulating the network cross-linking chemistry. Common cross-linking strategies rely on (i) irreversible network bond degradation and reformation in response to an external stimulus, (ii) using dynamic covalent chemistry, or (iii) isomerization of integrated functional groups (e.g., azobenzene or spiropyran). Many of these strategies are executed using ultraviolet or visible light since the incident photons serve as an external stimulus that affords spatial and temporal control over the mechanical adaptation process. Here, we describe a different type of hydrogel cross-linking strategy that uses a redox-responsive cross-linker, incorporated in poly(hydroxyethyl acrylate)-based hydrogels at three different weight percent loadings, which consists of two viologen subunits tethered by hexaethylene glycol and capped with styrene groups at each terminus. These dicationic viologen subunits (V2+) can be reduced to their monoradical cations (Vâ¢+) through a photoinduced electron transfer (PET) process using a visible light-absorbing photocatalyst (tris(bipyridine)ruthenium(II) dichloride) embedded in the hydrogel, resulting in the intramolecular stacking of viologen radical cations, through radical-radical pairing interactions, while losing two positive charges and the corresponding counteranions from the hydrogel. It is shown how this concerted process ultimately leads to collapse of the hydrogel network and significantly (p < 0.05) increases (by nearly a factor of 2) the soft material's stiffness, tensile strength, and percent elongation at break, all of which is easily reversed via oxidation of the viologen subunits and swelling in water. Application of this reversible PET process was demonstrated by photopatterning the same hydrogel multiple times, where the pattern was "erased" each time by turning off the blue light (â¼450 nm) source and allowing for oxidation and reswelling in between patterning steps. The areas of the hydrogel that were masked exhibited lower (by 1-2 kPa) shear storage moduli (G') than the areas that were irradiated for 1.5 h. Moreover, because the viologen subunits in the functional cross-linker are electrochromic, it is possible to visualize the regions of the hydrogel that undergo changes in mechanical properties. This visualization process was illustrated by photopatterning a larger hydrogel (â¼9.5 cm on its longest side) with a photomask in the design of an array of stars.
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Osteoarthritis (OA) is a debilitating joint disease resulting from chronic joint inflammation and erosion of articular cartilage. A promising biological treatment for OA is intra-articular administration of platelet-rich plasma (PRP). However, immediate bolus release of growth factors limits beneficial therapeutic effects of PRP, thus necessitating the demand for sustained release platforms. In this study, we evaluated the therapeutic value of PRP released from a polyethylene glycol (PEG) hydrogel on articular chondrocytes/cartilage explants derived from OA patients. Lyophilized PRP (PRGF) was encapsulated in PEG hydrogels at 10% w/v and hydrogel swelling, storage modulus and degradation and PRGF release kinetics were determined. PRGF releasate from the hydrogels was collected on day 1, 4, and 11. Encapsulation of PRGF at 10% w/v in PEG hydrogels had minimal effect on hydrogel properties. PRGF was released with an initial burst followed by sustained release until complete hydrogel degradation. Effect of PRGF releasates and bolus PRGF (1% w/v PRGF) on patient-derived cartilage explants or chondrocytes was assessed by chondrocyte proliferation (pico-green assay), gene expression for COL1A1, COL2A1, MMP13, COX2, and NFKB1 (real-time polymerase chain reaction), and measurement of nitric oxide concentration (Griess' assay). Compared to bolus PRGF, PRGF releasates enhanced chondrocyte proliferation, suppressed the expression of genes like MMP13, NFKB1, COL1A1, and COL2A1 and reduced levels of nitric oxide. Taken together, these results indicate that release of PRGF from PEG hydrogels may improve the therapeutic efficacy of PRP and merits further investigation in an animal model of OA. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2401-2410, 2019.
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Condrócitos/fisiologia , Osteoartrite/terapia , Plasma Rico em Plaquetas , Cartilagem Articular/metabolismo , Proliferação de Células , Expressão Gênica , Glicosaminoglicanos/biossíntese , Humanos , Hidrogéis , Óxido Nítrico/biossíntese , Polietilenoglicóis , Cultura Primária de CélulasRESUMO
Honey is well-known for its wound healing capability and Manuka honey (MH) contains a unique Manuka factor, providing an additional antibacterial agent. Previously, there has not been a practical way to apply MH to a wound site, which renders treatment for an extended period extremely difficult. Tissue-engineered scaffolds offer an alternative treatment method to standard dressings by providing varying geometries to best treat the specific tissue. MH was incorporated into cryogels, hydrogels, and electrospun scaffolds to assess the effect of scaffold geometry on bacterial clearance and adhesion, as well as cellular adhesion. Electrospun scaffolds exhibited a faster release due to the nanoporous fibrous geometry which led to a larger partial bacterial clearance as compared to the more three-dimensional cryogels (CG) and hydrogels (HG). Similarly, the fast release of MH from the electrospun scaffolds resulted in reduced bacterial adhesion. Overall, the fast MH release of the electrospun scaffolds versus the extended release of the HG and CG scaffolds provides differences in cellular/bacterial adhesion and advantages for both short and long-term applications, respectively. This manuscript provides a comparison of the scaffold pore structures as well as bacterial and cellular properties, providing information regarding the relationship between varying scaffold geometry and MH efficacy.
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Mast cells have functional plasticity affected by their tissue microenvironment, which greatly impacts their inflammatory responses. Because lactic acid (LA) is abundant in inflamed tissues and tumors, we investigated how it affects mast cell function. Using IgE-mediated activation as a model system, we found that LA suppressed inflammatory cytokine production and degranulation in mouse peritoneal mast cells, data that were confirmed with human skin mast cells. In mouse peritoneal mast cells, LA-mediated cytokine suppression was dependent on pH- and monocarboxylic transporter-1 expression. Additionally, LA reduced IgE-induced Syk, Btk, and ERK phosphorylation, key signals eliciting inflammation. In vivo, LA injection reduced IgE-mediated hypothermia in mice undergoing passive systemic anaphylaxis. Our data suggest that LA may serve as a feedback inhibitor that limits mast cell-mediated inflammation.
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Anafilaxia/prevenção & controle , Anti-Inflamatórios não Esteroides/farmacologia , Retroalimentação Fisiológica , Imunoglobulina E/genética , Ácido Láctico/farmacologia , Mastócitos/efeitos dos fármacos , Tirosina Quinase da Agamaglobulinemia/genética , Tirosina Quinase da Agamaglobulinemia/imunologia , Anafilaxia/induzido quimicamente , Anafilaxia/imunologia , Anafilaxia/patologia , Animais , Dinitrofenóis/administração & dosagem , Dinitrofenóis/antagonistas & inibidores , Feminino , Regulação da Expressão Gênica , Cetoprofeno/farmacologia , Ácido Láctico/imunologia , Ácido Láctico/metabolismo , Mastócitos/imunologia , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/imunologia , Cavidade Peritoneal/patologia , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Albumina Sérica/administração & dosagem , Albumina Sérica/antagonistas & inibidores , Transdução de Sinais , Pele/efeitos dos fármacos , Pele/imunologia , Pele/patologia , Quinase Syk/genética , Quinase Syk/imunologia , Simportadores/genética , Simportadores/imunologiaRESUMO
Manuka honey is an ancient remedy to improve wound healing; however, an effective delivery system is needed to facilitate extended release of honey into wounds. We developed an electrospun dermal regeneration template consisting of a poly (ε-caprolactone) (PCL) scaffold embedded with 1%, 5%, 10%, or 20% manuka honey. In vitro studies demonstrated that honey PCL scaffolds were not toxic to macrophages, and they allowed for macrophage infiltration into the scaffolds. Vascular endothelial growth factor (VEGF), a marker of angiogenesis, was released by macrophages cultured with scaffolds and macrophage/scaffold conditioned media promoted endothelial cell tube formation in an angiogenesis assay. In a full thickness murine wound model, the scaffolds prevented rapid wound contraction. In vivo, cells infiltrated the scaffolds by post-wounding day 7, but the honey scaffolds did not affect collagen deposition at that time. In summary, preliminary studies investigating the effect of honey on tissue repair show that scaffolds prevent rapid wound contraction, allow for cell infiltration, and promote angiogenesis. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B:2620-2628, 2019.
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Mel , Macrófagos/metabolismo , Alicerces Teciduais/química , Cicatrização , Ferimentos e Lesões/terapia , Animais , Feminino , Humanos , Macrófagos/patologia , Camundongos , Ferimentos e Lesões/metabolismo , Ferimentos e Lesões/patologiaRESUMO
The aim of this study is to evaluate a core-shell nanofiber as a useful matrix for tuning Rosuvastatin (RSV) release and osteogenic differentiation in vitro. Polyvinyl alcohol (PVA) and silk fibroin were used as the shell and the core, respectively. To obtain a linear and beadless core-shell structure and an optimal release profile, the shell/core flow rate ratio was varied (0, 0.4, 0.6, 0.8, and 1). Formation of continuous nanofibers with an obvious core/sheath structure was proved using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Differential scanning calorimetry indicated the presence of two distinct phase structures in the nanofibers. Also, RSV molecules were dispersed in an amorphous state in the nanofibers. The in vitro release profile of the core-shell structure exhibited a biphasic release profile and the amount of released RSV was controlled by adjusting the shell flow rate. Human adipose-derived stem cells cultured on the RSV loaded nanofibers were found to improve cell proliferation and assist osteogenic differentiation as revealed by Alizarin red staining and real-time RT-PCR.
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Tecido Adiposo/citologia , Fibroínas/química , Nanofibras/química , Osteogênese/efeitos dos fármacos , Álcool de Polivinil/química , Rosuvastatina Cálcica/farmacologia , Células-Tronco/citologia , Engenharia Tecidual/métodos , Adulto , Animais , Bombyx , Varredura Diferencial de Calorimetria , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Liberação Controlada de Fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Cinética , Pessoa de Meia-Idade , Nanofibras/ultraestrutura , Osteogênese/genética , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , MolhabilidadeRESUMO
Volumetric muscle loss (VML) is a loss of over â¼10% of muscle mass that results in functional impairment. Although skeletal muscle possesses the ability to repair and regenerate itself following minor injuries, VML injuries are irrecoverable. Currently, there are no successful clinical therapies for the treatment of VML. Previous studies have treated VML defects with decellularized extracellular matrix (D-ECM) scaffolds derived from either pig urinary bladder or small intestinal submucosa. These therapies were unsuccessful due to the poor mechanical stability of D-ECM leading to quick degradation in vivo. To circumvent these issues, in this manuscript aligned nanofibers of D-ECM were created using electrospinning that mimicked native muscle architecture and provided topographical cues to primary satellite cells. Additionally, combining D-ECM with polycaprolactone (PCL) improved the tensile mechanical properties of the electrospun scaffold. In vitro testing shows that the electrospun scaffold with aligned nanofibers of PCL and D-ECM supports satellite cell growth, myogenic protein expression, and myokine production.
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Matriz Extracelular/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Nanofibras/química , Alicerces Teciduais/química , Animais , Anisotropia , Bovinos , Proliferação de Células , Células Cultivadas , DNA/análise , Masculino , Poliésteres , Ratos , Ratos Endogâmicos Lew , Regeneração , Estresse Mecânico , Resistência à Tração , Engenharia Tecidual/métodosRESUMO
Skeletal muscle is inept in regenerating after traumatic injuries due to significant loss of basal lamina and the resident satellite cells. To improve regeneration of skeletal muscle, we have developed biomimetic sponges composed of collagen, gelatin, and laminin (LM)-111 that were crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC). Collagen and LM-111 are crucial components of the muscle extracellular matrix and were chosen to impart bioactivity whereas gelatin and EDC were used to provide mechanical strength to the scaffold. Morphological and mechanical evaluation of the sponges showed porous structure, water-retention capacity and a compressive modulus of 590-808 kPa. The biomimetic sponges supported the infiltration and viability of C2 C12 myoblasts over 5 days of culture. The myoblasts produced higher levels of myokines such as VEGF, IL-6, and IGF-1 and showed higher expression of myogenic markers such as MyoD and myogenin on the biomimetic sponges. Biomimetic sponges implanted in a mouse model of volumetric muscle loss (VML) supported satellite, endothelial, and inflammatory cell infiltration but resulted in limited myofiber regeneration at 2 weeks post-injury. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 92-103, 2019.
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Materiais Biomiméticos , Músculo Esquelético , Mioblastos Esqueléticos , Regeneração/efeitos dos fármacos , Ferimentos e Lesões , Animais , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Linhagem Celular , Masculino , Camundongos , Músculo Esquelético/lesões , Músculo Esquelético/patologia , Músculo Esquelético/fisiologia , Mioblastos Esqueléticos/metabolismo , Mioblastos Esqueléticos/patologia , Porosidade , Ferimentos e Lesões/tratamento farmacológico , Ferimentos e Lesões/metabolismo , Ferimentos e Lesões/patologiaRESUMO
The current gold standard treatment for oral clefts is autologous bone grafting. This treatment, however, presents another wound site for the patient, greater discomfort, and pediatric patients have less bone mass for bone grafting. A potential alternative treatment is the use of tissue engineered scaffolds. Hydrogels are well characterized nanoporous scaffolds and cryogels are mechanically durable, macroporous, sponge-like scaffolds. However, there has been limited research on these scaffolds for cleft craniofacial defects. 3D-printed molds can be combined with cryogel/hydrogel fabrication to create patient-specific tissue engineered scaffolds. By combining 3D-printing technology and scaffold fabrication, we were able to create scaffolds with the geometry of three cleft craniofacial defects. The scaffolds were then characterized to assess the effect of the mold on their physical properties. While the scaffolds were able to completely fill the mold, creating the desired geometry, the overall volumes were smaller than expected. The cryogels possessed porosities ranging from 79.7% to 87.2% and high interconnectivity. Additionally, the cryogels swelled from 400% to almost 1500% of their original dry weight while the hydrogel swelling did not reach 500%, demonstrating the ability to fill a defect site. Overall, despite the complex geometry, the cryogel scaffolds displayed ideal properties for bone reconstruction.
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We present an insert-based approach to fabricate scalable and multiplexable microfluidic devices for 3D cell culture and integration with downstream detection modules. Laser-cut inserts with a layer of electrospun fibers are used as a scaffold for 3D cell culture, with the inserts being easily assembled in a 3D-printed fluidic device for flow-based studies. With this approach, the number and types of cells (on the inserts) in one fluidic device can be customized. Moreover, after an investigation (i.e., stimulation) under flowing conditions, the cell-laden inserts can be removed easily for subsequent studies including imaging and cell lysis. In this paper, we first discuss the fabrication of the device and characterization of the fibrous inserts. Two device designs containing two (channel width = 260 µm) and four (channel width = 180 µm) inserts, respectively, were used for different experiments in this study. Cell adhesion on the inserts with flowing media through the device was tested by culturing endothelial cells. Macrophages were cultured and stimulated under different conditions, the results of which indicate that the fibrous scaffolds under flow conditions result in dramatic effects on the amount and kinetics of TNF-α production (after LPS stimulation). Finally, we show that the cell module can be integrated with a downstream absorbance detection scheme. Overall, this technology represents a new and versatile way to culture cells in a more in vivo fashion for in vitro studies with online detection modules. Graphical abstract This paper describes an insert-based microfluidic device for 3D cell culture that can be easily scaled, multiplexed, and integrated with downstream analytical modules.