Dependence of crack shape in loaded articular cartilage on the collagenous structure.

Cartilage cracks disrupt tissue mechanics, alter cell mechanobiology, and often trigger tissue degeneration. Yet, some tissue cracks heal spontaneously. A primary factor determining the fate of tissue cracks is the compression-induced mechanics, specifically whether a crack opens or closes when loaded. Crack deformation is thought to be affected by tissue structure, which can be probed by quantitative polarized light microscopy (PLM). It is unclear how the PLM measures are related to deformed crack morphology. Here, we investigated the relationship between PLM-derived cartilage structure and mechanical behavior of tissue cracks by testing if PLM-derived structural measures correlated with crack morphology in mechanically indented cartilages. METHODS: Knee joint cartilages harvested from mature and immature animals were used for their distinct collagenous fibrous structure and composition. The cartilages were cut through thickness, indented over the cracked region, and processed histologically. Sample-specific birefringence was quantified as two-dimensional (2D) maps of azimuth and retardance, two measures related to local orientation and degree of alignment of the collagen fibers, respectively. The shape of mechanically indented tissue cracks, measured as depth-dependent crack opening, were compared with azimuth, retardance, or "PLM index," a new parameter derived by combining azimuth and retardance. RESULTS: Of the three parameters, only the PLM index consistently correlated with the crack shape in immature and mature tissues. CONCLUSION: In conclusion, we identified the relative roles of azimuth and retardance on the deformation of tissue cracks, with azimuth playing the dominant role. The applicability of the PLM index should be tested in future studies using naturally-occurring tissue cracks.

Deformation behaviors and mechanical impairments of tissue cracks in immature and mature cartilages.

Degeneration of articular cartilage is often triggered by a small tissue crack. As cartilage structure and composition change with age, the mechanics of cracked cartilage may depend on the tissue age, but this relationship is poorly understood. Here, we investigated cartilage mechanics and crack deformation in immature and mature cartilage exposed to a full-thickness tissue crack using indentation testing and histology, respectively. When a cut was introduced, tissue cracks opened wider in the mature cartilage compared to the immature cartilage. However, the opposite occurred upon mechanical indentation over the cracked region. Functionally, the immature-cracked cartilages stress-relaxed faster, experienced increased tissue strain, and had reduced instantaneous stiffness, compared to the mature-cracked cartilages. Taken together, mature cartilage appears to withstand surface cracks and maintains its mechanical properties better than immature cartilage and these superior properties can be explained by the structure of their collagen fibrous network.

Application of "Magnetic Anchors" to Align Collagen Fibres for Axonal Guidance.

The use of neural scaffolds with a highly defined microarchitecture, fabricated with standard techniques such as electrospinning and microfluidic spinning, requires surgery for their application to the site of injury. To circumvent the risk associated with aciurgy, new strategies for treatment are sought. This has led to an increase in the quantity of research into injectable hydrogels in recent years. However, little research has been conducted into controlling the building blocks within these injectable hydrogels to produce similar scaffolds with a highly defined microarchitecture. "Magnetic particle string" and biomimetic amphiphile self-assembly are some of the methods currently available to achieve this purpose. Here, we developed a "magnetic anchor" method to improve the orientation of collagen fibres within injectable 3D scaffolds. This procedure uses GMNP (gold magnetic nanoparticle) "anchors" capped with CMPs (collagen mimetic peptides) that "chain" them to collagen fibres. Through the application of a magnetic field during the gelling process, these collagen fibres are aligned accordingly. It was shown in this study that the application of CMP functionalised GMNPs in a magnetic field significantly improves the alignment of the collagen fibres, which, in turn, improves the orientation of PC12 neurites. The growth of these neurite extensions, which were shown to be significantly longer, was also improved. The PC12 cells grown in collagen scaffolds fabricated using the "magnetic anchor" method shows comparable cellular viability to that of the untreated collagen scaffolds. This capability of remote control of the alignment of fibres within injectable collagen scaffolds opens up new strategic avenues in the research for treating debilitating neural tissue pathologies.

Polycaprolactone-Based Scaffolds Facilitates Osteogenic Differentiation of Human Adipose-Derived Stem Cells in a Co-Culture System.

(1) Background: Stem cells in combination with scaffolds and bioactive molecules have made significant contributions to the regeneration of damaged bone tissues. A co-culture system can be effective in enhancing the proliferation rate and osteogenic differentiation of the stem cells. Hence, the aim of this study was to investigate the osteogenic differentiation of human adipose derived stem cells when co-cultured with human osteoblasts and seeded on polycaprolactone (PCL):hydroxyapatite (HA) scaffold; (2) Methods: Human adipose-derived stem cells (ASC) and human osteoblasts (HOB) were seeded in three different ratios of 1:2, 1:2 and 2:1 in the PCL-HA scaffolds. The osteogenic differentiation ability was evaluated based on cell morphology, proliferation rate, alkaline phosphatase (ALP) activity, calcium deposition and osteogenic genes expression levels using quantitative RT-PCR; (3) Results: The co-cultured of ASC/HOB in ratio 2:1 seeded on the PCL-HA scaffolds showed the most positive osteogenic differentiation as compared to other groups, which resulted in higher ALP activity, calcium deposition and osteogenic genes expression, particularly Runx, ALP and BSP. These genes indicate that the co-cultured ASC/HOB seeded on PCL-HA was at the early stage of osteogenic development; (4) Conclusions: The combination of co-culture system (ASC/HOB) and PCL-HA scaffolds promote osteogenic differentiation and early bone formation.

Directing Axonal Growth: A Review on the Fabrication of Fibrous Scaffolds That Promotes the Orientation of Axons.

Tissues are commonly defined as groups of cells that have similar structure and uniformly perform a specialized function. A lesser-known fact is that the placement of these cells within these tissues plays an important role in executing its functions, especially for neuronal cells. Hence, the design of a functional neural scaffold has to mirror these cell organizations, which are brought about by the configuration of natural extracellular matrix (ECM) structural proteins. In this review, we will briefly discuss the various characteristics considered when making neural scaffolds. We will then focus on the cellular orientation and axonal alignment of neural cells within their ECM and elaborate on the mechanisms involved in this process. A better understanding of these mechanisms could shed more light onto the rationale of fabricating the scaffolds for this specific functionality. Finally, we will discuss the scaffolds used in neural tissue engineering (NTE) and the methods used to fabricate these well-defined constructs.

Mechanical compression controls the biosynthesis of human osteoarthritic chondrocytes in vitro.

BACKGROUND: Cartilage damage, which can potentially lead to osteoarthritis, is a leading cause of morbidity in the elderly population. Chondrocytes are sensitive to mechanical stimuli and their matrix-protein synthesis may be altered when chondrocytes experience a variety of in vivo loadings. Therefore, a study was conducted to evaluate the biosynthesis of isolated osteoarthritic chondrocytes which subjected to compression with varying dynamic compressive strains and loading durations. METHODS: The proximal tibia was resected as a single osteochondral unit during total knee replacement from patients (N = 10). The osteoarthritic chondrocytes were isolated from the osteochondral units, and characterized using reverse transcriptase-polymerase chain reaction. The isolated osteoarthritic chondrocytes were cultured and embedded in agarose, and then subjected to 10% and 20% uniaxial dynamic compression up to 8-days using a bioreactor. The morphological features and changes in the osteoarthritic chondrocytes upon compression were evaluated using scanning electron microscopy. Safranin O was used to detect the presence of cartilage matrix proteoglycan expression while quantitative analysis was conducted by measuring type VI collagen using an immunohistochemistry and fluorescence intensity assay. FINDINGS: Gene expression analysis indicated that the isolated osteoarthritic chondrocytes expressed chondrocyte-specific markers, including BGN, CD90 and HSPG-2. Moreover, the compressed osteoarthritic chondrocytes showed a more intense and broader deposition of proteoglycan and type VI collagen than control. The expression of type VI collagen was directly proportional to the duration of compression in which 8-days compression was significantly higher than 4-days compression. The 20% compression showed significantly higher intensity compared to 10% compression in 4- and 8-days. INTERPRETATION: The biosynthetic activity of human chondrocytes from osteoarthritic joints can be enhanced using selected compression regimes.

Mechanical Strain-Mediated Tenogenic Differentiation of Mesenchymal Stromal Cells Is Regulated through Epithelial Sodium Channels.

It has been suggested that mechanical strain may elicit cell differentiation in adult somatic cells through activation of epithelial sodium channels (ENaC). However, such phenomenon has not been previously demonstrated in mesenchymal stromal cells (MSCs). The present study was thus conducted to investigate the role of ENaC in human bone marrow-derived MSCs (hMSCs) tenogenic differentiation during uniaxial tensile loading. Passaged-2 hMSCs were seeded onto silicone chambers coated with collagen I and subjected to stretching at 1 Hz frequency and 8% strain for 6, 24, 48, and 72 hours. Analyses at these time points included cell morphology and alignment observation, immunocytochemistry and immunofluorescence staining (collagen I, collagen III, fibronectin, and N-cadherin), and gene expression (ENaC subunits, and tenogenic markers). Unstrained cells at similar time points served as the control group. To demonstrate the involvement of ENaC in the differentiation process, an ENaC blocker (benzamil) was used and the results were compared to the noninhibited hMSCs. ENaC subunits' (α, ß, γ, and δ) expression was observed in hMSCs, although only α subunit was significantly increased during stretching. An increase in tenogenic genes' (collagen1, collagen3, decorin, tenascin-c, scleraxis, and tenomodulin) and proteins' (collagen I, collagen III, fibronectin, and N-cadherin) expression suggests that hMSCs underwent tenogenic differentiation when subjected to uniaxial loading. Inhibition of ENaC function resulted in decreased expression of these markers, thereby suggesting that ENaC plays a vital role in tenogenic differentiation of hMSCs during mechanical loading.

A LEGO inspired fiber probe analytical platform for early diagnosis of Dengue fever.

Based on the concept of LEGO toys, a fiber probe analytical platform (FPAP) was developed as a powerful diagnostic tool offering higher sensitivity in detection of infectious agents compared to established methods. Using the form and the function of LEGO toys, this protocol describes a fiber-based, 96-well plate, which suspends a new class of chemically-designed, electrospun fibers within the assay. This clamping strategy allows both sides of the developed fiber mats to interact with biomolecules within the assay thus benefiting from the tailored chemical and physical properties of these fiber-based bioreceptors in attracting the biomolecules to the surface. The fabrication method of FPAP involves one-step electrospinning of the chemically designed fibers, 3D printing of the LEGO-like probing segments, and assembly of the device followed by ELISA procedure. FPAP follows the same principles of operation as that of a conventional enzyme linked immunosorbent assay (ELISA), therefore, it can be run by lab technicians, expert in ELISA. FPAP was used for early diagnosis of Dengue fever and provided an 8-fold higher sensitivity while the limit of detection (LOD) was recorded to be in femto-gram per milliliter range which is significantly low when compared to other existing techniques or conventional assay. This platform allows different types of paper/fiber bio-receptive platforms to be incorporated within the design that promises simultaneous recognition of multiple infectious agents.

Electrospin-Coating of Paper: A Natural Extracellular Matrix Inspired Design of Scaffold.

Paper has recently found widespread applications in biomedical fields, especially as an alternative scaffolding material for cell cultures, owing to properties such as its fibrous nature, porosity and flexibility. However, paper on its own is not an optimal material for cell cultures as it lacks adhesion moieties specific to mammalian cells, and modifications such as hydrogel integration and chemical vapor deposition are necessary to make it a favorable scaffolding material. The present study focuses on modification of filter paper through electrospin-coating and dip-coating with polycaprolactone (PCL), a promising biomaterial in tissue engineering. Morphological analysis, evaluation of cell viability, alkaline phosphatase (ALP) activity and live/dead assays were conducted to study the potential of the modified paper-based scaffold. The results were compared to filter paper (FP) and electrospun PCL (ES-PCL) as reference samples. The results indicate that electrospin-coating paper is a simple and efficient way of modifying FP. It not only improves the morphology of the deposited electrospun layer through reduction of the fiber diameter by nearly 75%, but also greatly reduces the scaffold fabrication time compared to ES-PCL. The biochemical assays (Resazurin and ALP) indicate that electrospin-coated filter paper (ES-PCL/FP) provides significantly higher readings compared to all other groups. The live/dead results also show improved cell-distribution and cell-scaffold attachment all over the ES-PCL/FP.

Uniaxial Cyclic Tensile Stretching at 8% Strain Exclusively Promotes Tenogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stromal Cells.

The present study was conducted to establish the amount of mechanical strain (uniaxial cyclic stretching) required to provide optimal tenogenic differentiation expression in human mesenchymal stromal cells (hMSCs) in vitro, in view of its potential application for tendon maintenance and regeneration. Methods. In the present study, hMSCs were subjected to 1 Hz uniaxial cyclic stretching for 6, 24, 48, and 72 hours; and were compared to unstretched cells. Changes in cell morphology were observed under light and atomic force microscopy. The tenogenic, osteogenic, adipogenic, and chondrogenic differentiation potential of hMSCs were evaluated using biochemical assays, extracellular matrix expressions, and selected mesenchyme gene expression markers; and were compared to primary tenocytes. Results. Cells subjected to loading displayed cytoskeletal coarsening, longer actin stress fiber, and higher cell stiffness as early as 6 hours. At 8% and 12% strains, an increase in collagen I, collagen III, fibronectin, and N-cadherin production was observed. Tenogenic gene expressions were highly expressed (p < 0.05) at 8% (highest) and 12%, both comparable to tenocytes. In contrast, the osteoblastic, chondrogenic, and adipogenic marker genes appeared to be downregulated. Conclusion. Our study suggests that mechanical loading at 8% strain and 1 Hz provides exclusive tenogenic differentiation; and produced comparable protein and gene expression to primary tenocytes.

Carpal Bone Segmentation Using Fully Convolutional Neural Network.

BACKGROUND: Bone Age Assessment (BAA) refers to a clinical procedure that aims to identify a discrepancy between biological and chronological age of an individual by assessing the bone age growth. Currently, there are two main methods of executing BAA which are known as Greulich-Pyle and Tanner-Whitehouse techniques. Both techniques involve a manual and qualitative assessment of hand and wrist radiographs, resulting in intra and inter-operator variability accuracy and time-consuming. An automatic segmentation can be applied to the radiographs, providing the physician with more accurate delineation of the carpal bone and accurate quantitative analysis. METHODS: In this study, we proposed an image feature extraction technique based on image segmentation with the fully convolutional neural network with eight stride pixel (FCN-8). A total of 290 radiographic images including both female and the male subject of age ranging from 0 to 18 were manually segmented and trained using FCN-8. RESULTS AND CONCLUSION: The results exhibit a high training accuracy value of 99.68% and a loss rate of 0.008619 for 50 epochs of training. The experiments compared 58 images against the gold standard ground truth images. The accuracy of our fully automated segmentation technique is 0.78 ± 0.06, 1.56 ±0.30 mm and 98.02% in terms of Dice Coefficient, Hausdorff Distance, and overall qualitative carpal recognition accuracy, respectively.

Structural and bone marrow stem cell biocompatibility studies of hydrogel synthesized via chemo-enzymatic route.

Natural biopolymers have many attractive medical applications; however, complications due to fibrosis caused a reduction in diffusion and dispersal of nutrients and waste products. Consequently, severe immunocompatibility problems and poor mechanical and degradation properties in synthetic polymers ensue. Hence, the present study investigates a novel hydrogel material synthesized from caprolactone, ethylene glycol, ethylenediamine, polyethylene glycol, ammonium persulfate, and tetramethylethylenediamine via chemo-enzymatic route. Spectroscopic analyses indicated the formation of polyurea and polyhydroxyurethane as the primary building block of the hydrogel starting material. Biocompatibility studies showed positive observation in biosafety test using direct contact cytotoxicity assay in addition to active cellular growth on the hydrogel scaffold based on fluorescence observation. The synthesized hydrogel also exhibited (self)fluorescence properties under specific wavelength excitation. Hence, synthesized hydrogel could be a potential candidate for medical imaging as well as tissue engineering applications as a tissue expander, coating material, biosensor, and drug delivery system.

Electrospin-coating of nitrocellulose membrane enhances sensitivity in nucleic acid-based lateral flow assay.

Point-of-care biosensors are important tools developed to aid medical diagnosis and testing, food safety and environmental monitoring. Paper-based biosensors, especially nucleic acid-based lateral flow assays (LFA), are affordable, simple to produce and easy to use in remote settings. However, the sensitivity of such assays to infectious diseases has always been a restrictive challenge. Here, we have successfully electrospun polycaprolactone (PCL) on nitrocellulose (NC) membrane to form a hydrophobic coating to reduce the flow rate and increase the interaction rate between the targets and gold nanoparticles-detecting probes conjugates, resulting in the binding of more complexes to the capture probes. With this approach, the sensitivity of the PCL electrospin-coated test strip has been increased by approximately ten-fold as compared to the unmodified test strip. As a proof of concept, this approach holds great potential for sensitive detection of targets at point-of-care testing.

Immobilisation of hydroxyapatite-collagen on polydopamine grafted stainless steel 316L: Coating adhesion and in vitro cells evaluation.

The utilisation of hydroxyapatite and collagen as bioactive coating materials could enhance cells attachment, proliferation and osseointegration. However, most methods to form crystal hydroxyapatite coating do not allow the incorporation of polymer/organic compound due to production phase of high sintering temperature. In this study, a polydopamine film was used as an intermediate layer to immobilise hydroxyapatite-collagen without the introduction of high sintering temperature. The surface roughness, coating adhesion, bioactivity and osteoblast attachment on the hydroxyapatite-collagen coating were assessed as these properties remains unknown on the polydopamine grafted film. The coating was developed by grafting stainless steel 316L disks with a polydopamine film. Collagen type I fibres were then immobilised on the grafted film, followed by the biomineralisation of hydroxyapatite. The surface roughness and coating adhesion analyses were later performed by using AFM instrument. An Alamar Blue assay was used to determine the cytotoxicity of the coating, while an alkaline phosphatase activity test was conducted to evaluate the osteogenic differentiation of human fetal osteoblasts on the coating. Finally, the morphology of cells attachment on the coating was visualised under FESEM. The highest RMS roughness and coating adhesion were observed on the hydroxyapatite-collagen coating (hydroxyapatite-coll-dopa). The hydroxyapatite-coll-dopa coating was non-toxic to the osteoblast cells with greater cells proliferation, greater level of alkaline phosphate production and more cells attachment. These results indicate that the immobilisation of hydroxyapatite and collagen using an intermediate polydopamine is identical to enhance coating adhesion, osteoblast cells attachment, proliferation and differentiation, and thus could be implemented as a coating material on orthopaedic and dental implants.

Electrospun Polycaprolactone Nanofibers as a Reaction Membrane for Lateral Flow Assay.

Electrospun polycaprolactone (PCL) nanofibers have emerged as a promising material in diverse biomedical applications due to their various favorable features. However, their application in the field of biosensors such as point-of-care lateral flow assays (LFA) has not been investigated. The present study demonstrates the use of electrospun PCL nanofibers as a reaction membrane for LFA. Electrospun PCL nanofibers were treated with NaOH solution for different concentrations and durations to achieve a desirable flow rate and optimum detection sensitivity in nucleic acid-based LFA. It was observed that the concentration of NaOH does not affect the physical properties of nanofibers, including average fiber diameter, average pore size and porosity. However, interestingly, a significant reduction of the water contact angle was observed due to the generation of hydroxyl and carboxyl groups on the nanofibers, which increased their hydrophilicity. The optimally treated nanofibers were able to detect synthetic Zika viral DNA (as a model analyte) sensitively with a detection limit of 0.5 nM. Collectively, the benefits such as low-cost of fabrication, ease of modification, porous nanofibrous structures and tunability of flow rate make PCL nanofibers a versatile alternative to nitrocellulose membrane in LFA applications. This material offers tremendous potential for a broad range of point-of-care applications.

Polymethacrylate Coated Electrospun PHB Fibers as a Functionalized Platform for Bio-Diagnostics: Confirmation Analysis on the Presence of Immobilized IgG Antibodies against Dengue Virus.

In this article, a combination of far field electrospinning (FFES) and free-radical polymerization has been used to create a unique platform for protein immobilization via the physical attachment of biomolecules to the surface of the fiber mats. The large specific surface area of the fibers with its tailored chemistry provides a desirable platform for effective analyte-surface interaction. The detailed analysis of protein immobilization on a newly developed bio-receptive surface plays a vital role to gauge its advantages in bio-diagnostic applications. We relied on scanning electron microscopy (SEM), diameter range analysis, and X-ray photoelectron spectroscopy (XPS), along with thermal gravimetric analysis (TGA), water-in-air contact angle analysis (WCA), Fourier transform infrared spectroscopy (FTIR), and atomic force microscopy (AFM) to study our developed platforms and to provide valuable information regarding the presence of biomolecular entities on the surface. Detailed analyses of the fiber mats before and after antibody immobilization have shown obvious changes on the surface of the bioreceptive surface including: (i) an additional peak corresponding to the presence of an antibody in TGA analysis; (ii) extra FTIR peaks corresponding to the presence of antibodies on the coated fiber platforms; and (iii) a clear alteration in surface roughness recorded by AFM analysis. Confirmation analyses on protein immobilization are of great importance as they underlay substantial grounds for various biosensing applications.

Development of poly (1, 8-octanediol citrate)/chitosan blend films for tissue engineering applications.

Blends of poly (1, 8-octanediol citrate) (POC) and chitosan (CS) were prepared through solution casting technique. Films with different component fractions (POC/CS: 100/0, 90/10, 80/20, 70/30, 60/40, and 0/100) were successfully prepared and characterized for their mechanical, thermal, structural and morphological properties as well as biocompatibility. The incorporation of CS to POC significantly increased tensile strength and elastic modulus and presented limited influences on pH variation which is important to the biocompatibility of biomaterial implants. The assessment of surface topography indicated that blending could enhance and control the surface roughness of the pure films. POC/CS blends well-supported human dermal fibroblast cells attachment and proliferation, and thus can be used for a range of tissue engineering applications.

Fate of tenogenic differentiation potential of human bone marrow stromal cells by uniaxial stretching affected by stretch-activated calcium channel agonist gadolinium.

The role for mechanical stimulation in the control of cell fate has been previously proposed, suggesting that there may be a role of mechanical conditioning in directing mesenchymal stromal cells (MSCs) towards specific lineage for tissue engineering applications. Although previous studies have reported that calcium signalling is involved in regulating many cellular processes in many cell types, its role in managing cellular responses to tensile loading (mechanotransduction) of MSCs has not been fully elucidated. In order to establish this, we disrupted calcium signalling by blocking stretch-activated calcium channel (SACC) in human MSCs (hMSCs) in vitro. Passaged-2 hMSCs were exposed to cyclic tensile loading (1 Hz + 8% for 6, 24, 48, and 72 hours) in the presence of the SACC blocker, gadolinium. Analyses include image observations of immunochemistry and immunofluorescence staining from extracellular matrix (ECM) production, and measuring related tenogenic and apoptosis gene marker expression. Uniaxial tensile loading increased the expression of tenogenic markers and ECM production. However, exposure to strain in the presence of 20 µM gadolinium reduced the induction of almost all tenogenic markers and ECM staining, suggesting that SACC acts as a mechanosensor in strain-induced hMSC tenogenic differentiation process. Although cell death was observed in prolonged stretching, it did not appear to be apoptosis mediated. In conclusion, the knowledge gained in this study by elucidating the role of calcium in MSC mechanotransduction processes, and that in prolonged stretching results in non-apoptosis mediated cell death may be potential useful for regenerative medicine applications.

Paper-based capacitive sensors for identification and quantification of chemicals at the point of care.

The identification and quantification of chemicals play a vital role in evaluation and surveillance of environmental health and safety. However, current techniques usually depend on costly equipment, professional staff, and/or essential infrastructure, limiting their accessibility. In this work, we develop paper-based capacitive sensors (PCSs) that allow simple, rapid identification and quantification of various chemicals from microliter size samples with the aid of a handheld multimeter. PCSs are low-cost parallel-plate capacitors (~$0.01 per sensor) assembled from layers of aluminum foil and filter paper via double-sided tape. The developed PCSs can identify different kinds of fluids (e.g., organic chemicals) and quantify diverse concentrations of substances (e.g., heavy metal ions) based on differences in dielectric properties, including capacitance, frequency spectrum, and dielectric loss tangent. The PCS-based method enables chemical identification and quantification to take place much cheaply, simply, and quickly at the point-of-care (POC), holding great promise for environmental monitoring in resource-limited settings.

Hypoxia enhances the viability, growth and chondrogenic potential of cryopreserved human adipose-derived stem cells.

Cryopreservation is the only existing method of storage of human adipose-derived stem cells (ASCs) for clinical use. However, cryopreservation has been shown to be detrimental to ASCs, particularly in term of cell viability. To restore the viability of cryopreserved ASCs, it is proposed to culture the cells in a hypoxic condition. To this end, we aim to investigate the effect of hypoxia on the cryopreserved human ASCs in terms of not only cell viability, but also their growth and stemness properties, which have not been explored yet. In this study, human ASCs were cultured under four different conditions: fresh (non-cryopreserved) cells cultured in 1) normoxia (21% O2) and 2) hypoxia (2% O2) and cryopreserved cells cultured in 3) normoxia and 4) hypoxia. ASCs at passage 3 were subjected to assessment of viability, proliferation, differentiation, and expression of stemness markers and hypoxia-inducible factor-1 alpha (HIF-1α). We found that hypoxia enhances the viability and the proliferation rate of cryopreserved ASCs. Further, hypoxia upregulates HIF-1α in cryopreserved ASCs, which in turn activates chondrogenic genes to promote chondrogenic differentiation. In conclusion, hypoxic-preconditioned cryopreserved ASCs could be an ideal cell source for cartilage repair and regeneration.