Cell-seeded Scaffolds Research Articles

Healthy but not osteoarthritic human meniscus-derived matrix scaffolds promote meniscus repair.

Meniscus tissue is commonly injured due to sports-related injuries and age-related degeneration and approximately 50% of individuals with a meniscus tear will develop post-traumatic osteoarthritis (PTOA). Given that the meniscus has limited healing potential, new therapeutic strategies are required to enhance meniscus repair. Porcine meniscus-derived matrix (MDM) scaffolds improve meniscus integrative repair, but sources of human meniscus tissue have not been investigated. Therefore, the objectives of this study were to generate healthy and osteoarthritic (OA) MDM scaffolds and to compare meniscus cellular responses and integrative repair. Meniscus cells showed high viability on both healthy and OA scaffolds. While DNA content was higher in cell-seeded OA scaffolds than cell-seeded healthy scaffolds, CCK-8, and both sGAG and collagen content were similar between scaffold types. After 28days in an ex vivo meniscus defect model, healthy and OA scaffolds had similar DNA, sGAG, and collagen content. However, the shear strength of repair was reduced in defects containing OA scaffolds compared to healthy scaffolds. In conclusion, healthy human allograft tissue is a useful source for generating MDM scaffolds that can support cellular growth, ECM production, and ex vivo integrative repair of the meniscus, highlighting the potential suitability for tissue engineering approaches to improve meniscus repair.

Reliable Fabrication of Mineral-Graded Scaffolds by Spin-Coating and Laser Machining for Use in Tendon-to-Bone Insertion Repair.

A reliable method for fabricating biomimetic scaffolds with a controllable mineral gradient to facilitate the surgical repair of tendon-to-bone injuries and the regeneration of the enthesis is reported. The gradient in mineral content is created by sequentially spin-coating with hydroxyapatite/poly(ε-caprolactone) suspensions containing hydroxyapatite nanoparticles in decreasing concentrations. To produce pores and facilitate cell infiltration, the spin-coated film is released and patterned with an array of funnel-shaped microchannels by laser machining. The unique design provided both mechanical (i.e., substrate stiffness) and biochemical (e.g., hydroxyapatite content) cues to spatially control the graded differentiation of mesenchymal stem cells. Immunocytochemical analysis of human mesenchymal stem cell-seeded scaffolds after 14 days of culture demonstrated the formation of a spatial phenotypic cell gradient from osteoblasts to mineralized chondrocytes based on the level of mineralization in the scaffold. By successfully recreating compositional and cellular features of the native tendon enthesis, the biomimetic scaffolds offer a promising avenue for improved tendon-to-bone repair.

Proteomic profiling of regenerated urinary bladder tissue in a non-human primate augmentation model

Urinary bladder dysfunction can be caused by environmental, genetic, and developmental insults. Depending upon insult severity, the bladder may lose its ability to maintain volumetric capacity and intravesical pressure resulting in renal deterioration. Bladder augmentation enterocystoplasty (BAE) is utilized to increase bladder capacity to preserve renal function using autologous bowel tissue as a “patch.” To avoid the clinical complications associated with this procedure, we have engineered composite grafts comprised of autologous bone marrow mesenchymal stem cells (MSCs) co-seeded with CD34+ hematopoietic stem/progenitor cells (HSPCs) onto a pliable synthetic scaffold [poly(1,8-octamethylene-citrate-co-octanol)(POCO)] or a biological scaffold (SIS; small intestinal submucosa) to regenerate bladder tissue in our baboon bladder augmentation model. We set out to determine the global protein expression profile of bladder tissue that has undergone regeneration with the aforementioned stem cell seeded scaffolds along with baboons that underwent BAE. Data demonstrate that POCO and SIS grafted animals share high protein homogeneity between native and regenerated tissues while BAE animals displayed heterogeneous protein expression between the tissues following long-term engraftment. We posit that stem cell-seeded scaffolds can recapitulate tissue that is nearly indistinguishable from native tissue at the protein level and may be used in lieu of procedures such as BAE.

The effect of Wharton’s jelly-derived stem cells seeded/boron-loaded acellular scaffolds on the healing of full-thickness burn wounds in the rat model

Burn wounds are the most destructive and complicated type of skin or underlying soft tissue injury that are exacerbated by a prolonged inflammatory response. Several cell-based therapeutic systems through the culturing of potent stem cells on modified scaffolds have been developed to direct the burn healing challenges. In this context, a new regenerative platform based on boron (B) enriched-acellular sheep small intestine submucosa (AOSIS) scaffold was designed and used as a carrier for mesenchymal stem cells derived from Wharton’s jelly (WJMSCs) aiming to promote the tissue healing in burn-induced rat models. hWJMSCs have been extracted from human extra-embryonic umbilical cord tissue. Thereafter, 96 third‐degree burned Wistar male rats were divided into 4 groups. The animals that did not receive any treatment were considered as group A (control). Then, group B was treated just by AOSIS scaffold, group C was received cell-seeded AOSIS scaffold (hWJMSCs-AOSIS), and group D was covered by boron enriched-cell-AOSIS scaffold (B/hWJMSCs-AOSIS). Inflammatory factors, histopathological parameters, and the expression levels of epitheliogenic and angiogenic proteins were assessed on 5, 14 and 21 d post-wounding. Application of the B/hWJMSCs-AOSIS on full-thickness skin-burned wounds significantly reduced the volume of neutrophils and lymphocytes at day 21 post-burning, whilst the number of fibroblasts and blood vessels enhanced at this time. In addition, molecular and histological analysis of wounds over time further verified that the addition of boron promoted wound healing, with decreased inflammatory factors, stimulated vascularization, accelerated re-epithelialization, and enhanced expression levels of epitheliogenic genes. In addition, the boron incorporation amplified wound closure via increasing collagen deposition and fibroblast volume and activity. Therefore, this newly fabricated hWJMSCs/B-loaded scaffold can be used as a promising system to accelerate burn wound reconstruction through inflammatory regulation and angiogenesis stimulation.

ELECTRICAL STIMULATION COMBINED WITH ADDITIVE MANUFACTURED 3D CONDUCTIVE SCAFFOLDS TOWARDS IMPROVED BONE-TISSUE ENGINEERING STRATEGIES

The growing number of non-union fractures in an aging population has increased the clinical demand for tissue-engineered bone. Electrical stimulation (ES) has been described as a promising strategy for bone regeneration treatments in several clinical studies. However the underlying mechanism by which ES augments bone formation is still poorly understood and its use in bone tissue engineering (BTE) strategies is currently underexplored. Additive manufacturing (AM) technologies (Fused Deposition Modeling/3D Printing) have been widely used in BTE due to their ability to fabricate scaffolds with a high control over their structural and mechanical properties in a reproducible and scalable manner. Thus, in this work, we combined AM methods with conductive biomaterials and ES to enhance the osteogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells (hBMSCs) envisaging improved BTE strategies.First, we started by developing AM-based electro-bioreactor devices containing medical-grade electrodes (stainless steel and Ti6Al4V) to apply ES to monolayer 2D cultures and 3D cell-seeded scaffolds. Computer modeling(Finite Element Analysis-FEA) was employed to predict the magnitude/distribution of electrical fields within the ES devices and along the different conductive scaffolds. Prior to scaffold culture, 5 different ES protocols were tested in terms of their ability to promote hBMSCs proliferation and osteogenic differentiation in 2D cultures. The best performance ES protocol was then used in two different AM-based BTE strategies: 1) Two different conductive scaffolds (conductive poly lactic acid (PLA) and titanium) were seeded with hBMSCs and cultured for 21 days under osteogenic medium conditions with and without ES and their biological performance was evaluated in comparison to non-conductive standard PLA scaffolds; 2) Different PEDOT:PSS-based coating solutions were screened to obtain PEDOT:PSS/Gelatin-coated 3D polycaprolactone (PCL) scaffolds with a high(11 S.cm-1) and stable electroconductivity. When cultured under ES, PEDOT:PSS/Gelatin-PCL scaffolds enhanced significantly hBMSCs osteogenic differentiation and mineralization(calcium deposition), highlighting their potential for BTE applications.Acknowledgements: Funding received from FCT through projects InSilico4OCReg (PTDC/EME-SIS/0838/2021), OptiBioScaffold (PTDC/EME-SIS/4446/2020) and BioMaterARISES (EXPL/CTM-CTM/0995/2021), and to the institutions iBB (UIDB/04565/2020), CDRSP (UIDB/04044/2020) and Associate Laboratory i4HB (LA/P/0140/2020).

In Vitro and In Vivo Evaluation of 3D Printed Poly(Ethylene Glycol) Dimethacrylate-Based Photocurable Hydrogel Platform for Bone Tissue Engineering.

This study focuses to develop a unique hybrid hydrogel bioink formulation that incorporates poly(ethylene glycol) dimethacrylate (PEGDMA), gelatin (Gel), and methylcellulose (MC). This formulation achieves the necessary viscosity for extrusion-based three-dimensional (3D) printing of scaffolds intended for bone regeneration. After thorough optimization of the hybrid bioink system with Gel, three distinct scaffold groups are investigated in vitro: 0%, 3%, and 6% (w/v) Gel. These scaffold groups are examined for their morphology, mechanical strength, biodegradation, in vitro cell proliferation and differentiation, and in vivo bone formation using a rat cranial defect model. Among these scaffold compositions, the 3% Gel scaffold exhibits the most favorable characteristics, prompting further evaluation as a rat mesenchymal stem cell (rMSC) carrier in a critical-size cranial defect within a Lewis rat model. The compressive strength of all three scaffold groups range between 1 and 2MPa. Notably, the inclusion of Gel in the scaffolds leads to enhanced bioactivity and cell adhesion. The Gel-containing scaffolds notably amplify osteogenic differentiation, as evidenced by alkaline phosphatase (ALP) and Western blot analyses. The in vivo results, as depicted by microcomputed tomography, showcase augmented osteogenesis within cell-seeded scaffolds, thus validating this innovative PEGDMA-based scaffold system as a promising candidate for cranial bone defect healing.

A biomimetic multi-layer scaffold with collagen and zinc doped bioglass as a skin-regeneration agent in full-thickness injuries and its effects in vitro and in vivo

Due to the multilayer structure of skin tissue, the fabrication of a 3-layer scaffold could result in planned dermal regeneration. Herein, polyurethane (PU) and polycaprolactone (PCL), as a function of their mechanical stability and collagen due to its arginine-glycine-aspartic acid sequences, zinc ions because of overcoming the common problems of biological factors were employed. The scaffolds' physical, mechanical, and biological properties were examined by SEM, FTIR, contact angle, mechanical tensile, bacteriocidal efficacy, and hemolysis. Also, after L-929 fibroblast seeding, their biological activity was determined by SEM, DAPI, and MTT assays. Then, the cell-seeded scaffolds were implanted in full-thickness wounds of rats and evaluated by wound closure, histological, and molecular techniques. The in vivo studies showed better wound closure with the composite scaffold containing zinc ions. While its dermal re-organization was retarded in the presence of zinc ions compared to the composite scaffold containing non-doped bioglass. Despite this, the doped composite scaffold indicated better observations with the histological evaluations than the nontreated and bare scaffold groups. Real-time PCR confirmed the higher expression of FGF2 and FGFR genes in rats treated with the zinc-doped composite scaffold. In conclusion, PU/PCL-collagen/PCL-collagen containing the doped or non-doped nanoparticles showed better potential to heal dermal injuries.

Tendon-derived matrix crosslinking techniques for electrospun multi-layered scaffolds.

Tendon tears are common and healing often occurs incompletely and by fibrosis. Tissue engineering seeks to improve repair, and one approach under investigation uses cell-seeded scaffolds containing biomimetic factors. Retention of biomimetic factors on the scaffolds is likely critical to maximize their benefit, while minimizing the risk of adverse effects, and without losing the beneficial effects of the biomimetic factors. The aim of the current study was to evaluate cross-linking methods to enhance the retention of tendon-derived matrix (TDM) on electrospun poly(ε-caprolactone) (PCL) scaffolds. We tested the effects of ultraviolet (UV) or carbodiimide (EDC:NHS:COOH) crosslinking methods to better retain TDM to the scaffolds and stimulate tendon-like matrix synthesis. Initially, we tested various crosslinking configurations of carbodiimide (2.5:1:1, 5:2:1, and 10:4:1 EDC:NHS:COOH ratios) and UV (30 s 1 J/cm2 , 60 s 1 J/cm2 , and 60 s 4 J/cm2 ) on PCL films compared to un-crosslinked TDM. We found that no crosslinking tested retained more TDM than coating alone (Kruskal-Wallis: p > .05), but that human adipose stem cells (hASCs) spread most on the 60 s 1 J/cm2 UV- and 2.5:1:1 EDC-crosslinked films (Kruskal-Wallis: p < .05). Next, we compared the effects of 60 s 1 J/cm2 UV- and 2.5:1:1 EDC-crosslinked to TDM-coated and untreated PCL scaffolds on hASC-induced tendon-like differentiation. UV-crosslinked scaffolds had greater modulus and stiffness than PCL or TDM scaffolds, and hASCs spread more on UV-crosslinked scaffolds (ANOVA: p < .05). Fourier transform infrared spectra revealed that UV- or EDC-crosslinking TDM did not affect the peaks at wavenumbers characteristic of tendon. Crosslinking TDM to electrospun scaffolds improves tendon-like matrix synthesis, providing a viable strategy for improving retention of TDM on electrospun PCL scaffolds.

Application of Menstrual Blood Derived Stromal (stem) Cells Exert Greater Regenerative Potency Than Fibroblasts/Keratinocytes in Chronic Wounds of Diabetic Mice.

In this study we differentially showed the effects of cell-seeded bilayer scaffold wound dressing in accelerating healing process in diabetic ulcers that still remains as a major clinical challenge. The aim of the study was to compare immunomodulatory and angiogenic activity, and regenerative effect differences between Menstrual blood-derived Stem Cells (MenSCs) and foreskin-derived keratinocytes/fibroblasts. The streptozotocin-induced diabetic mice model was developed in male C57/BL6 mice. A bilayer scaffold was fabricated by electrospining silk fibroin nano-fibers on human Amniotic Membrane (AM). Dermal fibroblasts and keratinocyte isolated from neonatal foreskin and MenSCs were isolated from the menstrual blood of healthy women. The diabetic mice were randomly divided into three groups including no treatment group, fibroblast/keratinocyte-seeded bilayer scaffold group (bSC+FK), and MenSCs-seeded bilayer scaffold group. The healing of full-thickness excisional wounds evaluations in the diabetic mice model in each group were evaluated at 3, 7, and 14 days after treatment. The gross and histological data sets significantly showed wound healing promotion via re-epithelialization and wound contraction along with enhanced regeneration in MenSCs-seeded bilayer scaffold group with the most similarity to adjacent intact tissue. Immunofluorescence staining of mouse skin depicted a descending trend of type III collagen along with the higher expression of involucrin as keratinocyte marker in the MenSCs-seeded bilayer nanofibrous scaffold group in comparison with other treatment groups from day 7 to day 14. Moreover, higher levels of CD31 and von Willebrand factor (VWF), and also a higher ratio of M2/M1 macrophages in association with higher levels of the neural marker were observed in the bSC+MenSCs group in comparison with bSC+FK and no treatment groups. Healing symptoms in wounds dressed with keratinocyte/fibroblast-seeded bilayer scaffold was significantly lower than MenSCs-seeded bilayer scaffold done on impaired diabetic wound chronicity.

Osteogenic Activity on NaOH-Etched Three-Dimensional-Printed Poly-ɛ-Caprolactone Scaffolds in Perfusion or Spinner Flask Bioreactor.

Bioreactor systems, for example, spinner flask and perfusion bioreactors, and cell-seeded three-dimensional (3D)-printed scaffolds are used in bone tissue engineering strategies to stimulate cells and produce bone tissue suitable for implantation into the patient. The construction of functional and clinically relevant bone graft using cell-seeded 3D-printed scaffolds within bioreactor systems is still a challenge. Bioreactor parameters, for example, fluid shear stress and nutrient transport, will crucially affect cell function on 3D-printed scaffolds. Therefore, fluid shear stress induced by spinner flask and perfusion bioreactors might differentially affect osteogenic responsiveness of pre-osteoblasts inside 3D-printed scaffolds. We designed and fabricated surface-modified 3D-printed poly-ɛ-caprolactone (PCL) scaffolds, as well as static, spinner flask, and perfusion bioreactors to determine fluid shear stress and osteogenic responsiveness of MC3T3-E1 pre-osteoblasts seeded on the scaffolds in the bioreactors using finite element (FE)-modeling and experiments. FE-modeling was used to quantify wall shear stress (WSS) distribution and magnitude inside 3D-printed PCL scaffolds within spinner flask and perfusion bioreactors. MC3T3-E1 pre-osteoblasts were seeded on NaOH surface-modified 3D-printed PCL scaffolds, and cultured in customized static, spinner flask, and perfusion bioreactors up to 7 days. The scaffolds' physicochemical properties and pre-osteoblast function were assessed experimentally. FE-modeling showed that spinner flask and perfusion bioreactors locally affected WSS distribution and magnitude inside the scaffolds. The WSS distribution was more homogeneous inside scaffolds in perfusion than in spinner flask bioreactors. The average WSS on scaffold-strand surfaces ranged from 0 to 6.5 mPa for spinner flask bioreactors, and from 0 to 4.1 mPa for perfusion bioreactors. Surface modification of scaffolds by NaOH resulted in a surface with a honeycomb-like pattern and increased surface roughness (1.6-fold), but decreased water contact angle (0.3-fold). Both spinner flask and perfusion bioreactors increased cell spreading, proliferation, and distribution throughout the scaffolds. Perfusion, but not spinner flask bioreactors more strongly enhanced collagen (2.2-fold) and calcium deposition (2.1-fold) throughout the scaffolds after 7 days compared with static bioreactors, likely due to uniform WSS-induced mechanical stimulation of the cells revealed by FE-modeling. In conclusion, our findings indicate the importance of using accurate FE models to estimate WSS and determine experimental conditions for designing cell-seeded 3D-printed scaffolds in bioreactor systems. Impact Statement The success of cell-seeded three-dimensional (3D)-printed scaffolds depends on cell stimulation by biomechanical/biochemical factors to produce bone tissue suitable for implantation into the patient. We designed and fabricated surface-modified 3D-printed poly-ɛ-caprolactone (PCL) scaffolds, as well as static, spinner flask, and perfusion bioreactors to determine wall shear stress (WSS) and osteogenic responsiveness of pre-osteoblasts seeded on the scaffolds using finite element (FE)-modeling and experiments. We found that cell-seeded 3D-printed PCL scaffolds within perfusion bioreactors more strongly enhanced osteogenic activity than within spinner flask bioreactors. Our results indicate the importance of using accurate FE-models to estimate WSS and determine experimental conditions for designing cell-seeded 3D-printed scaffolds in bioreactor systems.

EXPLOITING NOVEL 3D JELLYFISH COLLAGEN SPONGES FOR BONE TISSUE REGENERATION

Jellyfish collagens exhibit auspicious perspectives for tissue engineering applications primarily due to their outstanding compatibility with a wide range of cell types, low immunogenicity and biodegradability. Furthermore, derived from a non-mammalian source, jellyfish collagens reduce the risk of disease transmission, minimising therefore the ethical and safety concerns. The current study aims to investigate the potential of 3-dimensional jellyfish collagen sponges (3D-JCS) in promoting bone tissue regeneration.Both qualitative and quantitative analyses were performed in order to assess adhesion and proliferation of MC3T3 cells on 3D-JCL, as well as cell migration and bone-like ECM production. Histological and fluorescent dyes were used to stain mineral deposits (i.e. Alizarin Red S (ARS), Von Kossa, Tetracycline hydrochloride) while images were acquired using optical and confocal microscopy.Qualitative data indicated successful adhesion and proliferation of MC3T3 cells on the 3D-JCS as well as cell migration along with ECM production both on the inner and outer surface of the scaffolds. Moreover, quantitative analyses indicated a four-fold increase of ARS uptake between 2- and 3-dimensional cultures (N=3) as well as an eighteen-fold increase of ARS uptake for the 3D-JCS (N=3) when cultured in osteogenic conditions compared to control. This suggests the augmented osteogenic potential of MC3T3 cells when cultured on 3D-JCS. Nevertheless, the cell-mediated mineral deposition appeared to alter the mechanical properties of the jellyfish collagen sponges that were previously reported to exhibit low mechanical properties (compressive modulus: 1-2 kPa before culture).The biocompatibility, high porosity and pore interconnectivity of jellyfish collagen sponges promoted adhesion and proliferation of MC3T3 cells as well as cell migration and bone-like ECM production. Their unique features recommend the jellyfish collagen sponges as superior biomaterial scaffolds for bone tissue regeneration. Further studies are required to quantify the change in mechanical properties of the cell-seeded scaffolds and confirm their suitability for bone tissue regeneration. We predict that the 3D-JCS will be useful for future studies in both bone and bone-tendon interface regeneration.AcknowledgmentsThis research has been supported by a Medical Research Scotland Studentship award (ref: -50177-2019) in collaboration with Jellagen Ltd.

Ascorbic Acid 2-Phosphate-Releasing Supercritical Carbon Dioxide-Foamed Poly(L-Lactide-Co-epsilon-Caprolactone) Scaffolds Support Urothelial Cell Growth and Enhance Human Adipose-Derived Stromal Cell Proliferation and Collagen Production

Tissue engineering can provide a novel approach for the reconstruction of large urethral defects, which currently lacks optimal repair methods. Cell-seeded scaffolds aim to prevent urethral stricture and scarring, as effective urothelium and stromal tissue regeneration is important in urethral repair. In this study, the aim was to evaluate the effect of the novel porous ascorbic acid 2-phosphate (A2P)-releasing supercritical carbon dioxide-foamed poly(L-lactide-co-ε-caprolactone) (PLCL) scaffolds (scPLCLA2P) on the viability, proliferation, phenotype maintenance, and collagen production of human urothelial cell (hUC) and human adipose-derived stromal cell (hASC) mono- and cocultures. The scPLCLA2P scaffold supported hUC growth and phenotype both in monoculture and in coculture. In monocultures, the proliferation and collagen production of hASCs were significantly increased on the scPLCLA2P compared to scPLCL scaffolds without A2P, on which the hASCs formed nonproliferating cell clusters. Our findings suggest the A2P-releasing scPLCLA2P to be a promising material for urethral tissue engineering.

The use of autologous skeletal muscle-derived cells as a sling in the treatment of stress-induced urinary incontinence: An experimental study in dogs

PurposeThis is an experimental preclinical study testing the applicability of autologous skeletal muscle-derived cells as a treatment of SUI in a canine model.MethodsTen mongrel dogs were included in this study. Skeletal muscle was harvested for biopsy in 4 dogs. One month later, incontinence was induced in 8 dogs through urethrolysis. Biopsied muscle cells were incubated and expanded for 8 weeks. Muscle-derived cells were collected and covered with a polyglycolic acid (PGA) scaffold immersed in culture medium and coated with Matrigel to be used as a sling, which was placed suburethrally in 8 dogs; 4 had cell seeding, and 4 had scaffolds only. Urethral pressure (UP) measurements were performed at baseline and 2 &amp;amp; 6 weeks after sling insertion. The urethra was harvested 4 weeks after sling insertion for histopathology.ResultsOne month, a statistically significant increase of mean urethral pressure values compared to baseline was observed in all dogs with a scaffold inserted. The increase ranged from 5 to 40 cmH2O. Histopathology showed significant periurethral proliferation of skeletal muscles in 4 dogs with cell-seeded scaffolds. These levels were the maximum levels in dogs # 1 &amp;amp; 2. This was not the case in the 4 dogs that had slings only.ConclusionBased on the outcome of this preliminary experience, the use of skeletal muscle-seeded PGA scaffolds seems to be an easy and reproducible procedure which preserves histological differentiation and integrity in a canine model

Muscle-like Scaffolds for Biomechanical Stimulation in a Custom-Built Bioreactor.

Tissue engineering aims to develop in-vitro substitutes of native tissues. One approach of tissue engineering relies on using bioreactors combined with biomimetic scaffolds to produce study models or in-vitro substitutes. Bioreactors provide control over environmental parameters, place and hold a scaffold under desired characteristics, and apply mechanical stimulation to scaffolds. Polymers are often used for fabricating tissue-engineering scaffolds. In this study, polycaprolactone (PCL) collagen-coated microfilament scaffolds were cell-seeded with C2C12 myoblasts; then, these were grown inside a custom-built bioreactor. Cell attachment and proliferation on the scaffolds were investigated. A loading pattern was used for mechanical stimulation of the cell-seeded scaffolds. Results showed that the microfilaments provided a suitable scaffold for myoblast anchorage and that the custom-built bioreactor provided a qualified environment for the survival of the myoblasts on the polymeric scaffold. This PCL-based microfilament scaffold located inside the bioreactor proved to be a promising structure for the study of skeletal muscle models and can be used for mechanical stimulation studies in tissue engineering applications.

Fabrication and evaluation of poly (vinyl alcohol)/gelatin fibrous scaffold containing ZnO nanoparticles for skin tissue engineering applications

Skin engineering offers innovative constructs for the treatment of a wide range of skin diseases. Hybrid scaffolds were fabricated using poly (vinyl alcohol), gelatin and ZnO nanoparticles by electrospinning. Different concentrations of ZnO nanoparticles (0%, 0.5% and 1%) were incorporated into the PVA/gelatin scaffolds and the constructs were characterized by SEM, FTIR, XRD, contact angle, swelling, degradation, and biomechanical tests; the release behavior of nanoparticles from the scaffolds was also evaluated. In cellular studies, human mesenchymal stem cells (hu-MSCs) were extracted from the umbilical cord of a newborn baby and characterized by flow cytometry. Cell-seeded scaffolds were assessed by in vitro biocompatibility, SEM images and live/dead assay. Finally, the antibacterial activity of scaffolds against E. coli and S. aureus strain was determined. Due to the presence of the hydrophilic biopolymer of gelatin in the contact angle test, the scaffolds showed hydrophilic properties. With the addition of nanoparticles, the hydrophilicity, water absorption and degradation rates of scaffolds were somewhat reduced. No difference in tensile modulus was observed between scaffolds. The release rate of nanoparticles from scaffolds increased over time. The immunoprofile evaluation of extracted cells by flow cytometry technique confirmed the mesenchymal nature of the cells. Fluorescent staining showed that most cells were alive and had appropriate shape during six days of cell culture on PVA/gelatin/ZnO1wt% scaffold. All scaffolds had acceptable biocompatibility, but control and PVA/gelatin groups showed a higher cell survival rate. Scaffolds containing nanoparticles had a direct effect on inhibiting bacterial growth in a dose-dependent manner. Our findings suggest that composite scaffolds containing ZnO nanoparticles seeded with hu-MSCs might be suitable for promoting skin healing.

In Vitro Differentiation of Myoblast Cell Lines on Spider Silk Scaffolds in a Rotating Bioreactor for Vascular Tissue Engineering.

Functional construction of tissue-engineered vessels as an alternative to autologous vascular grafts has been shown to be feasible, however the proliferation of seeded smooth-muscle cells remains a limiting factor. We employed a rotating bioreactor system to improve myoblast cell differentiation on a spider silk scaffold for tissue-engineered vessel construction. C2C12 myofibroblast cells were seeded on the surface of spider silk scaffold constructs and cultivated in a rotating bioreactor system with a continuous rotation speed (1 rpm). Cell function, cell growth and morphological structure and expression of biomarkers were analyzed using scanning electron microscopy, the LIVE/DEAD® assay, Western blot and quantitative real-time PCR analyses. A dense myofibroblast cell sheet could be developed which resembled native blood vessel muscular tissue in morphological structure and in function. Bioreactor perfusion positively affected cell morphology, and increased cell viability and cell differentiation. The expression of desmin, MYF5 and MEF2D surged as an indication of myoblast differentiation. Cell-seeded scaffolds showed a tear-down at 18 N when strained at a set speed (20 mm min-1). Spider silk scaffolds appear to offer a reliable basis for engineered vascular constructs and rotating bioreactor cultivation may be considered an effective alternative to complex bioreactor setups to improve cell viability and biology.

Decellularized tilapia fish skin: A novel candidate for tendon tissue engineering.

The poor regenerative ability of injured tendon tissues remains a clinical challenge. However, decellularized extracellular matrix (ECM) combined with stem cells shows promise. In contrast to bovine and porcine ECM, marine-derived decellularized ECM has several advantages; it is easily obtained, poses less biological risk, and is not contraindicated on religious grounds. This study successfully fabricated decellularized tilapia fish skin (DTFS) with copious preserved collagen fibers and natural pore structures. The outer layer is smooth and dense, while the inner layer has a soft structure with a rough surface. After crosslinking with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS), crosslinked DTFS (C-DTFS) showed improved mechanics in dry and wet conditions. In vitro, the leach liquor of crosslinked DTFS showed no cytotoxicity and promoted migration and tenonic differentiation of tendon-derived stem cells (TDSCs). Meanwhile, TDSCs seeded in the inner surface of DTFS maintained viability, differentiated, and exhibited spreading. Furthermore, cell-seeded scaffolds guided the regeneration of tendon tissue in a rat Achilles tendon defect model. Our results suggest that DTFS combined with TDSCs is a novel and promising therapeutic option for tendon tissue engineering.

Administration Methods of Mesenchymal Stem Cells in the Treatment of Burn Wounds

Cellular therapies for burn wound healing, including the administration of mesenchymal stem or stromal cells (MSCs), have shown promising results. This review aims to provide an overview of the current administration methods in preclinical and clinical studies of bone-marrow-, adipose-tissue-, and umbilical-cord-derived MSCs for treating burn wounds. Relevant studies were identified through a literature search in PubMed and Embase and subjected to inclusion and exclusion criteria for eligibility. Additional relevant studies were identified through a manual search of reference lists. A total of sixty-nine studies were included in this review. Of the included studies, only five had clinical data from patients, one was a prospective case–control, three were case reports, and one was a case series. Administration methods used were local injection (41% in preclinical and 40% in clinical studies), cell-seeded scaffolds (35% and 20%), topical application (17% and 60%), and systemic injection (1% and 0%). There was great heterogeneity between the studies regarding experimental models, administration methods, and cell dosages. Local injection was the most common administration method in animal studies, while topical application was used in most clinical reports. The best delivery method of MSCs in burn wounds is yet to be identified. Although the potential of MSC treatment for burn wounds is promising, future research should focus on examining the effect and scalability of such therapy in clinical trials.

Development of fibroblast/endothelial cell-seeded collagen scaffolds for in vitro prevascularization.

The development of vascularized scaffolds remains one of the major challenges in tissue engineering, and co-culturing with endothelial cells is known as one of the possible approaches for this purpose. In this approach, optimization of cell culture conditions, scaffolds, and fabrication techniques is needed to develop tissue equivalents that will enable in vitro formation of a capillary network. Prevascularized equivalents will be more physiologically comparable to the native tissues and potentially prevent insufficient vascularization after implantation. This study aimed to culture human umbilical vein endothelial cells (HUVECs), alone or in co-culture with fibroblasts, on collagen scaffolds prepared by simple fabrication approaches for in vitro prevascularization. Different concentrations and ratios of HUVECs and fibroblasts seeded on collagen gel and sponge scaffolds under several culture conditions were examined. Cell viability, scaffolds morphology, and structure were analyzed. Collagen gel scaffolds showed good cell proliferation and viability, with higher proliferation rates for cells cultured in a 2:1 (fibroblasts: HUVECs) ratio and kept in endothelial cell growth medium. However, these matrices were unable to support endothelial cell sprouting. Collagen sponges were highly porous and showed good cell viability. However, they became fragile over time in culture, and they still lack signs of vascularization. Collagen scaffolds were a good platform for cell growth and viability. However, under the experimental conditions of this study, the HUVEC/fibroblast-seeded scaffolds were not suitable platforms to generate in vitro prevascularized equivalents. Our findings will be a valuable starting point to optimize culture microenvironments and scaffolds during fabrication of prevascularized scaffolds.

Uniparental parthenogenetic embryonic stem cell derivatives adaptable for bone and cartilage regeneration

Cells with the desired phenotype and number are critical for regenerative medicine and tissue engineering. Uniparental parthenogenetic embryonic stem cells (pESCs) share fundamental properties with embryonic stem cells. This study aims to determine the viability of pESC-based tissue engineering for bone and cartilage reconstruction. The mouse pESCs were cultured in suspension to form embryoid bodies. An adherent cultivation approach was employed to obtain parthenogenetic embryonic mesenchymal stem cells (pMSCs) from the embryoid bodies. Then, the pMSCs were cultured in conditional media to differentiate into osteogenic and chondrogenic lineages. The pESC-derived osteoblasts and chondroblasts were seeded into coral and sodium alginate scaffolds, respectively. The cell-seeded scaffolds were implanted into dorsal subcutaneous pockets of nude mice to evaluate ectopic reconstruction of bone and cartilage. We demonstrated that pESCs display the capacity to differentiate into all three germ layers. The generated pMSCs were able to differentiate into osteogenic and chondrogenic lineages, which survived well after seeding into coral and alginate acid scaffolds. Six weeks after cell-scaffold implantation, gross inspection and histological examination revealed that ectopic bone and cartilage tissues had successfully regenerated in the specimen. According to the findings of this study, pESC derivatives have a high potential for bone and cartilage regeneration.