Engineering of a decellularized bovine skin coated with antibiotics-loaded electrospun fibers with synergistic antibacterial activity for the treatment of infectious wounds.

An ideal antibacterial wound dressing with strong antibacterial behavior versus highly drug-resistant bacteria and great wound-healing capacity is still being developed. There is a clinical requirement to progress the current clinical cares that fail to fully restore the skin structure due to post-wound infections. Here, we aim to introduce a novel two-layer wound dressing using decellularized bovine skin (DBS) tissue and antibacterial nanofibers to design a bioactive scaffold with bio-mimicking the native extracellular matrix of both dermis and epidermis. For this purpose, polyvinyl alcohol (PVA)/chitosan (CS) solution was loaded with antibiotics (colistin and meropenem) and electrospun on the surface of the DBS scaffold to fabricate a two-layer antibacterial wound dressing (DBS-PVA/CS/Abs). In detail, the characterization of the fabricated scaffold was conducted using biomechanical, biological, and antibacterial assays. Based on the results, the fabricated scaffold revealed a homogenous three-dimensional microstructure with a connected pore network, a high porosity and swelling ratio, and favorable mechanical properties. In addition, according to the cell culture result, our fabricated two-layer scaffold surface had a good interaction with fibroblast cells and provided an excellent substrate for cell proliferation and attachment. The antibacterial assay revealed a strong antibacterial activity of DBS-PVA/CS/Abs against both standard strain and multidrug-resistant clinical isolates of Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli. Our bilayer antibacterial wound dressing is strongly suggested as an admirable wound dressing for the management of infectious skin injuries and now promises to advance with preclinical and clinical research.

The effect of source animal age, decellularization protocol, and sterilization method on bovine acellular dermal matrix as a scaffold for wound healing and skin regeneration.

BACKGROUND: Healing the full-thickness skin wounds has remained a challenge. One of the most frequently used grafts for skin regeneration is xenogeneic acellular dermal matrices (ADMs), including bovine ADMs. This study investigated the effect of the source animal age, enzymatic versus non-enzymatic decellularization protocols, and gamma irradiation versus ethylene oxide (EO) sterilization on the scaffold. METHODS: ADMs were prepared using the dermises of fetal bovine or calf skins. All groups were decellularized through chemical and mechanical methods, unless T-FADM samples, in which an enzymatic step was added to the decellularization protocol. All groups were sterilized with ethylene oxide (EO), except G-FADM which was sterilized using gamma irradiation. The scaffolds were characterized through scanning electron microscopy, differential scanning calorimetry, tensile test, MTT assay, DNA quantification, and real-time PCR. The performance of the ADMs in wound treatment was also evaluated macroscopically and histologically. RESULTS: All ADMs were effectively decellularized. In comparison to FADM (EO-sterilized fetal ADM), morphological, and mechanical properties of G-FADM, T-FADM, and CADM (EOsterilized calf ADM) were changed to different extents. In addition, the CADM and G-FADM were thermally more stable than the FADM and T-FADM. Although all ADMs were noncytotoxic, the wounds of the FADM, T-FADM, and G-FADM groups were contracted to almost 30.0% of the original area on day 7, significantly faster than the CADM (17.5% ± 1.7) and control (12.2% ± 1.59) groups. However, by day 21, all ADMs were mostly closed except for the untreated group (60.1 ± 1.8). CONCLUSION: Altogether, fetal source and EO-sterilized samples performed better than calf source and gamma-sterilized samples unless in some mechanical properties. There was no added value in using enzymatic treatment during the decellularization process. Our results suggest that the age, decellularization, and sterilization methods of animal source should be selected based on the clinical requirements.

Improved biological behaviours and osteoinductive capacity of the gelatin nanofibers while composites with GO/MgO.

Among the many polymers introduced for bone tissue engineering, natural polymers have more advantages due to their high biocompatibility and biodegradability, despite their low mechanical properties. Herein, gelatin nanofibers with and without magnesium oxide (MgO) and graphene oxide (GO) nanoparticles were fabricated by electrospinning. The fabricated gelatin and gelatin/GO/MgO nanofibers were examined using scanning electron microscopy, protein adsorption, cell attachment and viability assays. The results revealed that biological behaviours of the gelatin nanofibers significantly improved while incorporated with MgO and GO nanoparticles. In the following, osteosupportive capacity of the fabricated scaffolds was investigated by Alizarin-red staining, alkaline phosphatase activity, and calcium content, and bone-related gene and protein assays. The results revealed that the highest osteogenic differentiation potential of human-induced pluripotent stem cells (hiPSCs) was detected while these cells were cultured on the gelatin/GO/MgO nanofibers. However, these makers in the hiPSCs cultured on the gelatin nanofibers were also significantly increased in comparison with the cells cultured on the tissue culture plates as a control. In conclusion, the results revealed that predictable disadvantages in gelatin nanofibers can be greatly improved by the addition of MgO and GO nanoparticles, and the resulting composite scaffold could be a potential candidate for use in bone tissue engineering.

A novel hybrid polymer of PCL/fish gelatin nanofibrous scaffold improves proliferation and differentiation of Wharton's jelly-derived mesenchymal cells into islet-like cells.

BACKGROUND: Using a different source of stem cells to compensate for the lost beta cells is a promising way to cure diabetic patients. Besides, the best efficiency of insulin-producing cells (IPCs) will appear when we culture them in an environment similar to inside the body. Hence, three-dimensional (3D) culture ameliorates the differentiation of diverse kinds of stem cells into IPCs compared to those differentiated in two-dimensional (2D) culture. In this study, we aim to create an ideal differentiation environment by using PCL/Fish gelatin nanofibrous scaffolds to differentiate Wharton's jelly-derived mesenchymal cells (WJ-MSCs) to IPCs and compare them with a 2D cultured group. METHODS: The evaluation of cellular, molecular, and functional properties of differentiated cells on the 3D and 2D cultures was investigated by several assays such as electron microscopy, quantitative PCR, immunochemistry, western blotting, and ELISA. RESULTS: The in vitro studies showed that WJ-MSCs differentiated in the 3D culture have strong properties of IPCs such as islet-like cells. The expression of pancreatic-specific genes at both RNA and protein levels showed higher differentiation efficacy of 3D culture. Besides, the results of the ELISA tests demonstrate that in both groups the differentiated cells are functional and secreted C-peptide and insulin in glucose stimulation, but the secretion of C-peptide and insulin in the 3D culture group was higher than those cultured in 2D groups. CONCLUSION: Our findings showed the use of PCL/Fish gelatin nanofibrous scaffolds with optimized differentiation protocols can promote the differentiation of IPCs from WJ-MSCs compared to the 2D culture group.

Defining the role of 17ß-estradiol in human endometrial stem cells differentiation into neuron-like cells.

Human endometrial stem cells (hEnSCs) that can be differentiated into various neural cell types have been regarded as a suitable cell population for neural tissue engineering and regenerative medicine. Considering different interactions between hormones, growth factors, and other factors in the neural system, several differentiation protocols have been proposed to direct hEnSCs towards specific neural cells. The 17ß-estradiol plays important roles in the processes of development, maturation, and function of nervous system. In the present research, the impact of 17ß-estradiol (estrogen, E2) on the neural differentiation of hEnSCs was examined for the first time, based on the expression levels of neural genes and proteins. In this regard, hEnSCs were differentiated into neuron-like cells after exposure to retinoic acid (RA), epidermal growth factor (EGF), and also fibroblast growth factor-2 (FGF2) in the absence or presence of 17ß-estradiol. The majority of cells showed a multipolar morphology. In all groups, the expression levels of nestin, Tuj-1 and NF-H (neurofilament heavy polypeptide) (as neural-specific markers) increased during 14 days. According to the outcomes of immunofluorescence (IF) and real-time PCR analyses, the neuron-specific markers were more expressed in the estrogen-treated groups, in comparison with the estrogen-free ones. These findings suggest that 17ß-estradiol along with other growth factors can stimulate and upregulate the expression of neural markers during the neuronal differentiation of hEnSCs. Moreover, our findings confirm that hEnSCs can be an appropriate cell source for cell therapy of neurodegenerative diseases and neural tissue engineering.

Microengineered 3D cell-laden thermoresponsive hydrogels for mimicking cell morphology and orientation in cartilage tissue engineering.

Mimicking the zonal organization of native articular cartilage, which is essential for proper tissue functions, has remained a challenge. In this study, a thermoresponsive copolymer of chitosan-g-poly(N-isopropylacrylamide) (CS-g-PNIPAAm) was synthesized as a carrier of mesenchymal stem cells (MSCs) to provide a support for their proliferation and differentiation. Microengineered three-dimensional (3D) cell-laden CS-g-PNIPAAm hydrogels with different microstripe widths were fabricated to control cellular alignment and elongation in order to mimic the superficial zone of natural cartilage. Biochemical assays showed six- and sevenfold increment in secretion of glycosaminoglycans (GAGs) and total collagen from MSCs encapsulated within the synthesized hydrogel after 28 days incubation in chondrogenic medium. Chondrogenic differentiation was also verified qualitatively by histological and immunohistochemical assessments. It was found that 75 ± 6% of cells encapsulated within 50 µm wide microstripes were aligned with an aspect ratio of 2.07 ± 0.16 at day 5, which was more organized than those observed in unpatterned constructs (12 ± 7% alignment and a shape index of 1.20 ± 0.07). The microengineered constructs mimicked the cell shape and organization in the superficial zone of cartilage whiles the unpatterned one resembled the middle zone. Our results suggest that microfabrication of 3D cell-laden thermosensitive hydrogels is a promising platform for creating biomimetic structures leading to more successful multi-zonal cartilage tissue engineering. Biotechnol. Bioeng. 2017;114: 217-231. © 2016 Wiley Periodicals, Inc.

Preparation of bilayer tissue-engineered polyurethane/poly-L-lactic acid nerve conduits and their in vitro characterization for use in peripheral nerve regeneration.

BACKGROUND: Due to loss of peripheral nerve structure and/or function resulting from trauma, accidents, and other causes, peripheral nerve injuries continue to be a major clinical problem. These injuries can cause partial or total loss of sensory, motor, and autonomic capabilities as well as neuropathic pain. PNI affects between 13 and 23 out of every 100,000 people annually in developed countries. Regeneration of damaged nerves and restoration of function after peripheral nerve injury remain significant therapeutic challenges. Although autologous nerve graft transplantation is a viable therapy option in several clinical conditions, donor site morbidity and a lack of donor tissue often hinder full functional recovery. Biomimetic conduits used in tissue engineering to encourage and direct peripheral nerve regeneration by providing a suitable microenvironment for nerve ingrowth are only one example of the cutting-edge methods made possible by this field. Many innate extracellular matrix (ECM) structures of different tissues can be successfully mimicked by nanofibrous scaffolds. Nanofibrous scaffolds can closely mimic the surface structure and morphology of native ECMs of many tissues. METHODS: In this study, we have produced bilayer nanofibrous nerve conduit based on poly-lactic acid/polyurethane/multiwall carbon nanotube (PLA/PU/MWCNT), for application as composite scaffolds for static nerve tissue engineering. The contact angle was indicated to show the hydrophilicity properties of electrospun nanofibers. The SEM images were analyzed to determine the fiber's diameters, scaffold morphology, and endometrial stem cell adhesion. Moreover, MTT assay and DAPI staining were used to show the viability and proliferation of endometrial stem cells. RESULTS: The constructed bilayer PLA/PU/MWCNT scaffolds demonstrated the capacity to support cell attachment, and the vitality of samples was assessed using SEM, MTT assay, and DAPI staining technique. CONCLUSIONS: According to an in vitro study, electrospun bilayer PLA/PU/MWCNT scaffolds can encourage the adhesion and proliferation of human endometrial stem cells (hEnSCs) and create the ideal environment for increasing cell survival.

Luteolin-incorporated fish collagen hydrogel scaffold: An effective drug delivery strategy for wound healing.

In clinical practice, wound care has always been challenging. Hydrogels play a key role in facilitating active wound recovery by absorbing exudates, maintaining moisture, and alleviating pain through cooling. In this study, type I collagen was isolated from the skin of crucian carp (Carassius carassius) and verified by amino acid analysis, FTIR, and SDS-PAGE. By adopting a new approach, luteolin was added to collagen hydrogels in situ after being dissolved in an alkaline solution. XRD and SEM confirmed the luteolin was incorporated and entirely distributed throughout the hydrogel. The plastic compression improved the young's modulus of hydrogel to 15.24 ± 0.59 kPa, which is adequate for wound protection. The drug loading efficiency was 98 ± 1.47 % in the selected formulation. The luteolin-incorporated hydrogel enabled regulated drug release. We assessed the cytotoxicity using MTT and live-dead assays, as well as examined the hemocompatibility to determine the biocompatibility of the hydrogel. In vivo experiments showed that the hydrogel with luteolin had the highest wound closure rate (94.01 ± 2.1 %) and improved wound healing with granular tissue formation, collagen deposition, and re-epithelialization. These findings indicate that this efficient drug delivery technology can accelerate the process of wound healing.

Collagen short nanofiber-embedded chondroitin sulfate-hyaluronic acid nanocomposite: A cartilage-mimicking in situ-forming hydrogel with fine-tuned properties.

In situ-forming hydrogels that possess the ability to be injected in a less invasive manner and mimic the biochemical composition and microarchitecture of the native cartilage extracellular matrix are desired for cartilage tissue engineering. Besides, gelation time and stiffness of the hydrogel are two interdependent factors that affect cells' distribution and fate and hence need to be optimized. This study presented a bioinspired in situ-forming hydrogel composite of hyaluronic acid (HA), chondroitin sulfate (CS), and collagen short nanofiber (CSNF). HA and CS were functionalized with aldehyde and amine groups to form a gel through a Schiff-base reaction. CSNF was fabricated via electrospinning, followed by fragmentation by ultrasonics. Gelation time (11-360 s) and compressive modulus (1.4-16.2 kPa) were obtained by varying the concentrations of CS, HA, CSNFs, and CSNFs length. The biodegradability and biocompatibility of the hydrogels with varying gelation and stiffness were also assessed in vitro and in vivo. At three weeks, the assessment of hydrogels' chondrogenic differentiation also yields varying levels of chondrogenic differentiation. The subcutaneous implantation of the hydrogels in a mouse model indicated no severe inflammation. Results demonstrated that the injectable CS/HA@CSNF hydrogel was a promising hydrogel for tissue engineering and cartilage regeneration.

Fabrication of 3D chitosan/polyvinyl alcohol/brushite nanofibrous scaffold for bone tissue engineering by electrospinning using a novel falling film collector.

Despite its advantages, electrospinning has limited effectiveness in 3D scaffolding due to the high density of fibers it produces. In this research, a novel electrospinning collector was developed to overcome this constraint. An aqueous suspension containing chitosan/polyvinyl alcohol nanofibers was prepared employing a unique falling film collector. Suspension molding by freeze-drying resulted in a 3D nanofibrous scaffold (3D-NF). The mineralized scaffold was obtained by brushite deposition on 3D-NF using wet chemical mineralization by new sodium tripolyphosphate and calcium chloride dihydrate precursors. The 3D-NF was optimized and compared with the conventional electrospun 2D nanofibrous scaffold (2D-NF) and the 3D freeze-dried scaffold (3D-FD). Both minor fibrous and major freeze-dried pore shapes were present in 3D-NFs with sizes of 16.11-24.32 µm and 97.64-234.41 µm, respectively. The scaffolds' porosity increased by 53 % to 73 % compared to 2D-NFs. Besides thermal stability, mineralization improved the 3D-NF's ultimate strength and elastic modulus by 2.2 and 4.7 times, respectively. In vitro cell studies using rat bone marrow mesenchymal cells confirmed cell infiltration up to 290 µm and scaffold biocompatibility. The 3D-NFs given nanofibers and brushite inclusion exhibited considerable osteoinductivity. Therefore, falling film collectors can potentially be applied to prepare 3D-NFs from electrospinning without post-processing.

The Effect of Fetal Bovine Acellular Dermal Matrix Seeded with Wharton's Jelly Mesenchymal Stem Cells for Healing Full-Thickness Skin Wounds.

The treatment of full-thickness skin wounds is a problem in the clinical setting, as they do not heal spontaneously. Extensive pain at the donor site and a lack of skin grafts limit autogenic and allogeneic skin graft availability. We evaluated fetal bovine acellular dermal matrix (FADM) in combination with human Wharton's jelly mesenchymal stem cells (hWJ-MSCs) to heal full-thickness skin wounds. FADM was prepared from a 6-month-old trauma-aborted fetus. WJ-MSCs were derived from a human umbilical cord and seeded on the FADM. Rat models of full-thickness wounds were created and divided into three groups: control (no treatment), FADM, and FADM-WJMSCs groups. Wound treatment was evaluated microscopically and histologically on days 7, 14, and 21 post-surgery. The prepared FADM was porous and decellularized with a normal range of residual DNA. WJ-MSCs were seeded and proliferated on FADM effectively. The highest wound closure rate was observed in the FADM-WJMSC group on days 7 and 14 post-surgery. Furthermore, this group had fewer inflammatory cells than other groups. Finally, in this study, we observed that, without using the differential cell culture media of fibroblasts, the xenogeneic hWJSCs in combination with FADM could promote an increased rate of full-thickness skin wound closure with less inflammation.

Mesenchymal Stromal Cells and their EVs as Potential Leads for SARSCoV2 Treatment.

In December 2019, a betacoronavirus was isolated from pneumonia cases in China and rapidly turned into a pandemic of COVID-19. The virus is an enveloped positive-sense ssRNA and causes a severe respiratory syndrome along with a cytokine storm, which is the main cause of most complications. Therefore, treatments that can effectively control the inflammatory reactions are necessary. Mesenchymal Stromal Cells and their EVs are well-known for their immunomodulatory effects, inflammation reduction, and regenerative potentials. These effects are exerted through paracrine secretion of various factors. Their EVs also transport various molecules such as microRNAs to other cells and affect recipient cells' behavior. Scores of research and clinical trials have indicated the therapeutic potential of EVs in various diseases. EVs also seem to be a promising approach for severe COVID-19 treatment. EVs have also been used to develop vaccines since EVs are biocompatible nanoparticles that can be easily isolated and engineered. In this review, we have focused on the use of Mesenchymal Stromal Cells and their EVs for the treatment of COVID-19, their therapeutic capabilities, and vaccine development.

Injectable hydrogels in central nervous system: Unique and novel platforms for promoting extracellular matrix remodeling and tissue engineering.

Repairing central nervous system (CNS) is difficult due to the inability of neurons to recover after damage. A clinically acceptable treatment to promote CNS functional recovery and regeneration is currently unavailable. According to recent studies, injectable hydrogels as biodegradable scaffolds for CNS tissue engineering and regeneration have exceptionally desirable attributes. Hydrogel has a biomimetic structure similar to extracellular matrix, hence has been considered a 3D scaffold for CNS regeneration. An interesting new type of hydrogel, injectable hydrogels, can be injected into target areas with little invasiveness and imitate several aspects of CNS. Injectable hydrogels are being researched as therapeutic agents because they may imitate numerous properties of CNS tissues and hence reduce subsequent injury and regenerate neural tissue. Because of their less adverse effects and cost, easier use and implantation with less pain, and faster regeneration capacity, injectable hydrogels, are more desirable than non-injectable hydrogels. This article discusses the pathophysiology of CNS and the use of several kinds of injectable hydrogels for brain and spinal cord tissue engineering, paying particular emphasis to recent experimental studies.

The differentiation and generation of glucose-sensitive beta like-cells from menstrual blood-derived stem cells using an optimized differentiation medium with platelet-rich plasma (PRP).

Regarding their reversible damage of insulin-producing cells (IPCs) and the inefficiency of treatment methods for type 1 diabetes mellitus (T1DM), scientists decided to produce IPCs from an unlimited source of cells. But the production of these cells is constantly faced with problems such as low differentiation efficiency in cell therapy and regenerative medicine. This study provided an ideal differentiation medium enriched with plasma-rich platelet (PRP) delivery to produce IPCs from menstrual blood-derived stem cells (MenSCs). We compared them with and without PRP differentiation medium. MenSCs were then cultured in two experimental groups: with/without PRP differentiation medium and a control group (undifferentiated MenSCs). After 18 days, differentiated cells were analyzed for expression of pancreatic gene markers by real-time PCR. Immunocytochemical staining was used to detect the presence of insulin and Pdx-1 in the differentiated cells, and insulin and C-peptide secretion response to glucose were tested by ELISA. Finally, the morphology of differentiated cells was examined by an inverted microscope. In vitro studies showed that MenSCs differentiated in the PRP differentiation medium had strong properties of IPCs such as pancreatic islet-like structure. The expression of pancreatic markers at both RNA and protein levels showed that the differentiation efficiency was higher in the PRP differentiation medium. In both experimental groups, the differentiated cells were functional and secreted C-peptide and insulin on glucose stimulation, but the secretion of C-peptide and insulin in the PRP group was higher than those cultured in the without PRP differentiation medium. Our findings showed that using of PRP enriched differentiation medium can promote the differentiation of MenSCs into IPCs compared to the without PRP culture group. Therefore, the use of PRP into differentiation media can be proposed as a new approach to producing IPCs from MenSCs and used in cell-based therapies for T1DM.

Injectable nanocomposite hydrogels as an emerging platform for biomedical applications: A review.

Hydrogels have attracted much attention for biomedical and pharmaceutical applications due to the similarity of their biomimetic structure to the extracellular matrix of natural living tissues, tunable soft porous microarchitecture, superb biomechanical properties, proper biocompatibility, etc. Injectable hydrogels are an exciting type of hydrogels that can be easily injected into the target sites using needles or catheters in a minimally invasive manner. The more comfortable use, less pain, faster recovery period, lower costs, and fewer side effects make injectable hydrogels more attractive to both patients and clinicians in comparison to non-injectable hydrogels. However, it is difficult to achieve an ideal injectable hydrogel using just a single material (i.e., polymer). This challenge can be overcome by incorporating nanofillers into the polymeric matrix to engineer injectable nanocomposite hydrogels with combined or synergistic properties gained from the constituents. This work aims to critically review injectable nanocomposite hydrogels, their preparation methods, properties, functionalities, and versatile biomedical and pharmaceutical applications such as tissue engineering, drug delivery, and cancer labeling and therapy. The most common natural and synthetic polymers as matrices together with the most popular nanomaterials as reinforcements, including nanoceramics, carbon-based nanostructures, metallic nanomaterials, and various nanosized polymeric materials, are highlighted in this review.

Smart Carriers for Controlled Drug Delivery: Thermosensitive Polymers Embedded in Ordered Mesoporous Carbon.

An efficient drug delivery system was introduced. The carrier was synthesized by combination of an ordered mesoporous carbon (CMK3) and a thermosensitive polymer, poly(N-isopropylacrylamide), known as PNIPAAm. The polymers with 2 different chain lengths (PNIPAAm-100n and PNIPAAm-400n) were synthesized and each of the polymers was embedded in CMK3 to form composite materials. Nitrogen adsorption isotherm and scanning electron microscopy of the samples showed a uniform embedding of PNIPAAm-100n but a nonuniform embedding of PNIPAAm-400n. The latter observation is attributed to large intramolecular interactions of PNIPAAm-400n and their aggregation on the external surface of the porous structure. Doxorubicin was used as the model drug and was loaded onto the samples. The ultimate loading capacities for the polymer-embedded samples were reduced. However, the loading rates and the release capacities were significantly improved. Thermosensitivity of the polymer was introduced as the governing drug release mechanism; regardless of the polymer chain length, drug release at 37°C was significantly higher than 4°C. Cytotoxicity results confirmed materials' biocompatibility for future biological tests. It is clearly shown that the properly synthesized composite of ordered mesoporous carbon and thermosensitive polymer can be used as an efficient carrier for drug loading and release experiments. The loading and release profiles can be controlled by tailoring the polymer chain length.

Influence of polymer molecular weight on the in vitro cytotoxicity of poly (N-isopropylacrylamide).

Poly (N-isopropylacrylamide) (PNIPAAm) is a thermosensitive polymer with various biomedical applications. We examined molecular weight (MW)-dependent cytotoxicity of PNIPAAm. Our results indicated that low-MW PNIPAAm (degree of polymerization (DP)=35) is inherently toxic to cells. Moderate-MW PNIPAAms with their DP between 100 and 200 are non-cytotoxic. When cells are seeded on top of a polymer-coated surface, PNIPAAm with a higher MW (DP=400) shows non/low cytotoxicity, while when monolayer cells are exposed to the polymer solution, cell viability drops drastically. This may be due to lack of nutrient and oxygen rather than intrinsic toxicity of the polymer.

Poly(N-isopropylacrylamide) hydrogel/chitosan scaffold hybrid for three-dimensional stem cell culture and cartilage tissue engineering.

Providing a controllable and definable three-dimensional (3D) microenvironment for chondrogenic differentiation of mesenchymal stem cells (MSCs) remains a great challenge for cartilage tissue engineering. In this work, poly(N-isopropylacrylamide) (PNIPAAm) polymers with the degrees of polymerization of 100 and 400 (NI100 and NI400) were prepared and the polymer solutions were introduced into the preprepared chitosan porous scaffolds (CS) to form hybrids (CSNI100 and CSNI400, respectively). SEM images indicated that the PNIPAAm gel partially occupied chitosan pores while the interconnected porous structure of chitosan was preserved. MSCs were incorporated within the hybrid and cell proliferation and chondrogenic differentiation were monitored. After 7-day incubation of the cell-laden constructs in a growth medium, the cell viability in CSNI100 and CSNI400 were 54 and 108% higher than that in CS alone, respectively. Glycosaminoglycan and total collagen contents increased 2.6- and 2.5-fold after 28-day culture of cell-laden CSNI400 in the chondrogenic medium. These results suggest that the hybrid structure composed of the chitosan porous scaffold and the well-defined PNIPAAm hydrogel, in particular CSNI400, is suitable for 3D stem cell culture and cartilage tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2764-2774, 2016.