MACS-W:A modified optical clearing agent for imaging 3D cell cultures

Three-dimensional(3D)cell cultures have contributed to a variety of biological research fields by filling the gap between monolayers and animal models.The modern optical sectioning microscopic methods make it possible to probe the complexity of 3D cell cultures but are limited by the inherent opaqueness.While tissue optical clearing methods have emerged as powerful tools for investigating whole-mount tissues in 3D,they often have limitations,such as being too harsh for fragile 3D cell cultures,requiring complex handling protocols,or inducing tissue deformation with shrinkage or expansion.To address this issue,we proposed a modified optical clearing method for 3D cell cultures,called MACS-W,which is simple,highly efficient,and morphology-preserving.In our evaluation of MACS-W,we found that it exhibits excellent clearing capability in just 10 min,with minimal deformation,and helps drug evaluation on tumor spheroids.In summary,MACS-W is a fast,minimally-deformative and fluorescence compatible clearing method that has the potential to be widely used in the studies of 3D cell cultures.

ERK phosphorylation functions in invadopodia formation in tongue cancer cells in a novel silicate fibre-based 3D cell culture system

To screen for additional treatment targets against tongue cancer, we evaluated the contributions of extracellular signal-related kinase(ERK), AKT and ezrin in cancer development. Immunohistochemical staining showed that ERK and ezrin expressions were significantly higher in invasive squamous cell carcinoma than in carcinoma in situ. To investigate the roles of ERK and ezrin in cancer development, we used the non-woven silica fibre sheet Cellbedwith a structure resembling the loose connective tissue morphology in a novel 3 D culture system. We confirmed that the 3 D system using CellbedTMaccurately mimicked cancer cell morphology in vivo. Furthermore, cell projections were much more apparent in 3 D-cultured tongue cancer cell lines than in 2 D cultures. Typically, under conventional 2 D culture conditions, F-actin and cortactin are colocalized in the form of puncta within cells.However, in the 3 D-cultured cells, colocalization was mainly observed at the cell margins, including the projections. Projections containing F-actin and cortactin colocalization were predicted to be invadopodia. Although suppressing ezrin expression with small interfering RNA transfection caused no marked changes in morphology, cell projection formation was decreased, and the tumour thickness in vertical sections after 3 D culture was markedly decreased after suppressing ERK activity because both the invasion ability and proliferation were inhibited. An association between cortactin activation as well as ERK activity and invadopodia formation was detected. Our novel 3 D culture systems using Cellbed? are simple and useful for in vitro studies before conducting animal experiments. ERK contributes to tongue cancer development by increasing both cancer cell proliferation and migration via cortactin activation.

Matrix metalloproteinase-sensitive poly(ethylene glycol)/peptide hydrogels as an interactive platform conducive to cell proliferation during 3D cell culture

The response of extracellular matrix(ECM) to dynamic cell signals is of great significance for the regulation of cell behavior. In the present study, we prepared a type of matrix metalloproteinase(MMP)-sensitive degradable hydrogels(MSDHs) via the catalyst-free o-phthalaldehyde(OPA)/amine cross-linking reaction between o-phthalaldehyde-grafted four-arm poly(ethylene glycol)(4aPEG-OPA) and an MMP-sensitive degradable peptide. The gelation rates and storage moduli of MSDHs and the MMP-insensitive hydrogels(MIHs) based on an MMP-insensitive scramble peptide were comparable and dependent on the concentrations of precursor polymers. MSDHs were degradable while MIHs were stable in the presence of proteinase in vitro.The degradation of MSDHs was obviously faster than that of MIHs after subcutaneous injection into rats. In addition, both types of poly(ethylene glycol)/peptide hydrogels displayed excellent cytocompatibility in vitro, and showed good histocompatibility in vivo in the subcutaneous layer of rats. Furthermore, the proliferation of several MMP-expressing cell lines including MDA-MB-231 cells within MSDHs was obviously faster than that in MIHs, indicating the influence of metabolism-mediated scaffold degradation on the cell proliferation. This study provides a new biocompatible and biodegradable 3 D cell culture interactive platform for regulation of cell behavior.

Hydrogels with Dynamically Controllable Mechanics and Biochemistry for 3D Cell Culture Platforms

Many cell-matrix interaction studies have proved that dynamic changes in the extracellular matrix(ECM)are crucial to maintain cellular properties and behaviors.Thus,developing materials that can recapitulate the dynamic attributes of the ECM is highly desired for threedimensional(3 D)cell culture platforms.To this end,we sought to develop a hydrogel system that would enable dynamic and reversible turning of its mechanical and biochemical properties,thus facilitating the control of cell culture to imitate the natural ECM.Herein,a hydrogel with dynamic mechanics and a biochemistry based on an addition-fragmentation chain transfer(AFCT)reaction was constructed.Thiol-modified hyaluronic acid(HA)and allyl sulfide-modifiedε-poly-L-lysine(EPL)were synthesized to form hydrogels,which were non-swellable and biocompatible.The reversible modulus of the hydrogel was first achieved through the AFCT reaction;the modulus can also be regulated stepwise by changing the dose of UVA irradiation.Dynamic patterning of fluorescent markers in the hydrogel was also realized.Therefore,this dynamically controllable hydrogel has great potential as a 3 D cell culture platform for tissue engineering applications.

In Vitro Invasive Pattern of Hepatocellular Carcinoma Cell Line HCCLM9 Based on Three-dimensional Cell Culture and Quantum Dots Molecular Imaging

Summary： This study aimed to establish a new in vitro three-dimensional （3D） cell culture and use quantum dots （QDs） molecular imaging to examine the invasive behaviors of hepatocellular carcinoma （HCC） cells. Each well of the 24-well cell culture plate was cover-slipped. Matrigel diluted with se- rum-free DMEM was added and HCCLM9 cells were cultured on the Matrigel. The cell morphological and cell growth characteristics were observed by inverted microscopy and laser confocal microscopy at different culture time. Cell invasive features were monitored by QDs-based real-time molecular imaging techniques. The results showed that on this 3D cell culture platform, HCCLM9 cells exhibited typical multi-step invasive behaviors, including reversion of cell senescence, active focal proliferation and dominant clones invasion. During the process, cells under 3D cell culture showed biological behaviors of spatio-temporal characteristics. Cells first merged on the surface of matrix, then gradually infiltrated and migrated into deep part of matrix, presenting polygonal morphology with stretched protrusions, forming tubular, annular and even network structure, which suggested that HCC cells have the morpho- logical basis for vasculogenic mimicry. In addition, small cell clones with their edges well-circumscribed in early stage, progressed into a large irregular clone with ill-defined edge, while the other cells developed invadopodia. And QDs probing showed MT1-MMP was strongly expressed in the invadopodia. These findings indicate that a novel 3D cell culture platform has been successfully estab- lished, which can mimic the in vivo tumor microenvironment, and when combined with QDs-based mo- lecular imaging, it can help to better investigate the invasive behaviors of HCC cells.

Mechanical stretching of 3D hydrogels for neural stem cell differentiation

While it is known that mechanical dynamics are influential in neural differentiation for critical processes like neurogenesis or neurodegeneration, studies on neural stem cell therapies usually focus on biochemical interactions rather than mechanical aspects, frequently resulting in low efficacy and unfulfilled potential. Therefore, current studies are attempting to elucidate the effect of mechanical stimulus on neural performance using conventional two-dimensional(2D) planar substrates. Yet, these2D substrates fail to capture the defining three-dimensional(3D) characteristics of the in vivo neural stem cell environment.To complete this research gap, we synthesized a series of soft and elastic 3D hydrogels to mimic the neural tissue mechanical environment for 3D cell culture, using long-chain polyethylene glycol diacrylate(PEGDA) and gelatin-methacryloyl(Gel MA).By varying the concentration of the polymer, we obtained biomimicking hydrogels with a tensile modulus as low as 10 k Pa and a compressive modulus as low as 0.8 k Pa. The in vitro results demonstrated that Gel MA-PEGDA hydrogels have the high biocompatibility required to support neural cell growth, proliferation, and differentiation, as well as neurite outgrowth. We then studied the effect of mechanical stretching on the behaviors of neural cells and observed that mechanical stretching could significantly enhance neurite extension and axon elongation. In addition, the neurites were more directionally oriented to the stretching direction. Immunocytochemistry and relative gene expression data also suggested that mechanical tension could upregulate the expression of neural differentiation protein and genes, including GFAP and βIII-Tubulin. Overall, this study shows that in addition to the specific mechanical properties of Gel MA-PEGDA that improve neural differentiation towards specific lineages, hydrogel stretching is also a potentially attractive strategy to improve the therapeutic outcomes of neural stem cell therapies.

Breast Cancer MCF-7 Cell Spheroid Culture for Drug Discovery and Development

In vitro 3D cancer spheroids (tumoroids) exhibit a drug resistance profile similar to that found in solid tumors. 3D spheroid culture methods recreate more physiologically relevant microenvironments for cells. Therefore, these models are more appropriate for cancer drug screening. We have recently developed a protocol for MCF-7 cell spheroid culture, and used this method to test the effects of different types of drugs on this estrogen-dependent breast cancer cell spheroid. Our results demonstrated that MCF-7 cells can grow spheroid in medium using a low attachment plate. We managed to grow one spheroid in each well, and the spheroid can grow over a month, the size of the spheroid can grow over a hundred times in volume. Our targeted drug experimental results suggest that estrogen sulfotransferase, steroid sulfatase, and G protein-coupled estrogen receptor may play critical roles in MCF-7 cell spheroid growth, while estrogen receptors α and β may not play an essential role in MCF-7 spheroid growth. Organoids are the miniatures of in vivo tissues and reiterate the in vivo microenvironment of a specific organ, best fit for the in vitro studies of diseases and drug development. Tumoroid, developed from cancer cell lines or patients’ tumor tissue, is the best in vitro model of in vivo tumors. 3D spheroid technology will be the best future method for drug development of cancers and other diseases. Our reported method can be developed clinically to develop personalized drugs when the patient’s tumor tissues are used to develop a spheroid culture for drug screening.

Effects of simulated zero gravity on adhesion,cell structure,proliferation,and growth behavior,in glioblastoma multiforme

All life on Earth has evolved under the influence of continuous gravity,and methods have been developed to balance this influence with the biological evolution of organisms at the cellular and system levels.However,when exposed to zero gravity in space,the balance between cell structure and external forces is destroyed,resulting in changes at the cellular level(e.g.,cell morphology,adhesion,viability,apoptosis,etc.),and understanding the molecular mechanism of cell response to zero gravity will help to cope with diseases that rely on mechanical response.Therefore,biological research in space and zero gravity is a unique step in developing the best anti-cancer treatments,which is a great challenge to humanity.In this study,multicellular glioma cancer cells from a brain tumor in a 72-year-old Iraqi patient were subjected to simulated zero gravity for 24 h,and the results showed that most of the cells lost their adhesion,which is considered to be the first step toward cell apoptosis.In addition to the formation of multicellular spheroids,the results also showed that the inhibition rate for cell death was 32%in comparison to the control cells.Moreover,the cells showed a clear change in their cellular morphology and growth behavior.These results give new hope for fighting cancer distinctively,and such a treatment method has no side effects in comparison to traditional chemical and radiological ones.

Three-dimensional Culture of Human Airway Epithelium in Matrigel for Evaluation of Human Rhinovirus C and Bocavirus Infections

Objective Newly identified human rhinovirus C （HRV-C） and human bocavirus （HBoV） cannot propagate in vitro in traditional cell culture models; thus obtaining knowledge about these viruses and developing related vaccines are difficult. Therefore, it is necessary to develop a novel platform for the propagation of these types of viruses.Methods A platform for culturing human airway epithelia in a three-dimensional （3D） pattern using Matrigel as scaffold was developed. The features of 3D culture were identified by immunochemical staining and transmission electron microscopy. Nucleic acid levels of HRV-C and HBoV in 3D cells at designated time points were quantitated by real-time polymerase chain reaction {PCR）. Levels of cytokines, whose secretion was induced by the viruses, were measured by ELISA.Results Properties of bronchial-like tissues, such as the expression of biomarkers CK5, ZO-2, and PCK, and the development of cilium-like protuberances indicative of the human respiration tract, were observed in 3D-cultured human airway epithelial （HAE） cultures, but not in monolayer-cultured cells. Nucleic acid levels of HRV-C and HBoV and levels of virus-induced cytokines were also measured using the 3D culture system.Conclusion Our data provide a preliminary indication that the 3D culture model of primary epithelia using a Matrigel scaffold in vitro can be used to propagate HRV-C and HBoV.

In vitro 3D malignant melanoma model for the evaluation of hypericin-loaded oil-in-water microemulsion in photodynamic therapy

Advances in biomimetic three-dimensional(3D) melanoma models have brought new prospects of drug screening and disease modeling, since their physiological relevancy for recapitulating in vivo tumor architectures is more accurate than traditional two-dimensional(2D) cell culture. Gelatin methacryloyl(GelMA) is widely used as a tissue-engineered scaffold hydrogel for 3D cell culture. In the present study, an in vitro 3D malignant melanoma model based on Gel MA was fabricated to evaluate the efficiency of hypericin(Hy)-loaded microemulsion(ME) in photodynamic therapy against melanoma. The ME was produced by the spontaneous emulsification method to enhance the bioavailability of Hy at tumor sites. Hy-loaded MEs were applied to a 3D malignant melanoma model made using 6% Gel MA and the co-culture of B16F10 and Balb/c 3T3 cells,followed by crosslinking using violet light(403 nm). The observation revealed excellent cell viability and the presence of F-actin cytoskeleton network. Hy-loaded MEs exhibited higher phototoxicity and cell accumulation(about threefold) than free Hy, and the cells cultured in the 3D system displayed lower susceptibility(about 2.5-fold) than those in 2D culture.These findings indicate that the developed MEs are potential delivery carriers for Hy;furthermore, Gel MA hydrogel-based modeling in polydimethylsiloxane(PDMS) molds is a user-friendly and cost-effective in vitro platform to investigate drug penetration and provide a basis for evaluating nanocarrier efficiency for skin cancer and other skin-related diseases.

Sulfated GAG mimetic peptide nanofibers enhance chondrogenic differentiation of mesenchymal stem cells in 3D in vitro models

Articular cartilage,which is exposed to continuous repetitive compressive stress,has limited self-healing capacity in the case of trauma.Thus,it is crucial to develop new treatment options for the effective regeneration of the cartilage tissue.Current cellular therapy treatment options are microfracture and autologous chondrocyte implantation;however,these treatments induce the formation of fibrous cartilage,which degenerates over time,rather than functional hyaline cartilage tissue.Tissue engineering studies using biodegradable scaffolds and autologous cells are vital for developing an effective long-term treatment option.3D scaffolds composed of glycosaminoglycan-like peptide nanofibers are synthetic,bioactive,biocompatible,and biodegradable and trigger cell-cell interactions that enhance chondrogenic differentiation of cells without using any growth factors.We showed differentiation of mesenchymal stem cells into chondrocytes in both 2D and 3D culture,which produce a functional cartilage extracellular matrix,employing bioactive cues integrated into the peptide nanofiber scaffold without adding exogenous growth factors.

A micro-fragmented collagen gel as a cell-assembling platform for critical limb ischemia repair

Critical limb ischemia(CLI)is a devastating disease characterized by the progressive blockage of blood vessels.Although the paracrine effect of growth factors in stem cell therapy made it a promising angiogenic therapy for CLI,poor cell survival in the harsh ischemic microenvironment limited its efficacy.Thus,an imperative need exists for a stem-cell delivery method that enhances cell survival.Here,a collagen microgel(CMG)cell-delivery scaffold(40×20μm)was fabricated via micro-fragmentation from collagen-hyaluronic acid polyionic complex to improve transplantation efficiency.Culturing human adipose-derived stem cells(hASCs)with CMG enabled integrin receptors to interact with CMG to form injectable 3-dimensional constructs(CMG-hASCs)with a microporous microarchitecture and enhanced mass transfer.CMG-hASCs exhibited higher cell survival(p<0.0001)and angiogenic potential in tube formation and aortic ring angiogenesis assays than cell aggregates.Injection of CMG-hASCs intramuscularly into CLI mice increased blood perfusion and limb salvage ratios by 40%and 60%,respectively,compared to cell aggregate-treated mice.Further immunofluorescent analysis revealed that transplanted CMG-hASCs have greater muscle regenerative and angiogenic potential,with enhanced cell survival than cell aggregates(p<0.05).Collectively,we propose CMG as a cell-assembling platform and CMG-hASCs as promising therapeutics to treat CLI.

Mice 3D testicular organoid system as a novel tool to study Zika virus pathogenesis

Zika virus(ZIKV)poses a serious threat to global public health due to its close relationship with neurological and male reproductive damage.However,deficiency of human testicular samples hinders the in-depth research on ZIKV-induced male reproductive system injury.Organoids are relatively simple in vitro models,which could mimic the pathological changes of corresponding organs.In this study,we constructed a 3D testicular organoid model using primary testicular cells from adult BALB/c mice.Similar to the testis,this organoid system has a blood-testis barrier(BTB)-like structure and could synthesize testosterone.ZIKV tropism of testicular cells and ZIKV-induced pathological changes in testicular organoid was also similar to that in mammalian testis.Therefore,our results provide a simple and reproducible in vitro testicular model for the investigations of ZIKV-induced testicular injury.

自愈合导电复合水凝胶用于监测人体运动和3D细胞培养

导电水凝胶已在多个领域崭露头角,因为它具备将外界压力和形变转化为电信号的能力,可实现对物理和化学刺激的灵敏感知.然而,在导电水凝胶的研发中,需要克服平衡导电性和机械强度的挑战.本项工作中,为了提高导电水凝胶的力学性能,我们采用了多重策略.首先,引入了木质素-丹宁酸(lignin-TA)颗粒作为物理交联剂,同时充分利用羟丙基纤维素分子的多羟基结构,促进水凝胶半互穿聚合物网络的形成.随后,通过利用Fe^(3+)离子和TA分子之间的静电力和金属配位相互作用,进一步增强了聚合物网络之间的键合强度,提高了水凝胶的导电性.最终,所制备的导电水凝胶以其独特的网络结构,成功融合了物理和化学键的特性,并展现出卓越的自粘附、自愈合能力、电机械性能和生物相容性.这使其成为一个可用于监测人体活动和细胞增殖的理想材料.

Patient-derived organoids for therapy personalization in inflammatory bowel diseases

Inflammatory bowel diseases(IBDs)are chronic inflammatory disorders of the intestinal tract that have emerged as a growing problem in industrialized countries.Knowledge of IBD pathogenesis is still incomplete,and the most widely-accepted interpretation considers genetic factors,environmental stimuli,uncontrolled immune responses and altered intestinal microbiota composition as determinants of IBD,leading to dysfunction of the intestinal epithelial functions.In vitro models commonly used to study the intestinal barrier do not fully reflect the proper intestinal architecture.An important innovation is represented by organoids,3D in vitro cell structures derived from stem cells that can self-organize into functional organ-specific structures.Organoids may be generated from induced pluripotent stem cells or adult intestinal stem cells of IBD patients and therefore retain their genetic and transcriptomic profile.These models are powerful pharmacological tools to better understand IBD pathogenesis,to study the mechanisms of action on the epithelial barrier of drugs already used in the treatment of IBD,and to evaluate novel target-directed molecules which could improve therapeutic strategies.The aim of this review is to illustrate the potential use of organoids for therapy personalization by focusing on the most significant advances in IBD research achieved through the use of adult stem cells-derived intestinal organoids.

Using pipette tips to readily generate spheroids comprising single or multiple cell types

Three-dimensional(3D)cell culture methods have been validated that can replicate the tumor environment in vivo to a large extent,providing an effective tool for studying tumors.In this study,we demonstrated the use of standard laboratory pipette tips as micro vessels for generating 3D cell spheroids.No microfabrication or wet-chemistry surface modifications were involved in the procedure.Spheroids consisting of single or multiple cell types were generated within 24 h just by pipetting and incubating a cell suspension in pipette tips.Scanning electron microscope and optical microscope proved that the cells grew together tightly,and suggested that while gravity force might have initiated the sedimentation of cells at the bottom of the tip,the active aggregation of cells to form tight cell-cell interactions drove the formation of spheroids.Using common laboratory micropipettes and pipette tips,the rate of spheroid generation and the generation reproducibility was characterized from five boxes each with 80 tips.The ease of transferring reagents allowed modeling of the growth of microvascular endothelial cells in tumor spheroids.Moreover,the pairing and fusion of tumor spheroids could be manipulated in the pipette tips,suggesting the potential for building and assembling heterogeneous micro-tumor tissues in vitro to mimic solid tumors in vivo.This study demonstrated that spheroids can be readily and cost-effectively generated in standard biological laboratories in a timely manner using pipette tips.

Three-dimensional cell culture models for investigating human viruses

Three-dimensional(3D) culture models are physiologically relevant, as they provide reproducible results, experimental flexibility and can be adapted for high-throughput experiments. Moreover,these models bridge the gap between traditional two-dimensional(2D) monolayer cultures and animal models. 3D culture systems have significantly advanced basic cell science and tissue engineering, especially in the fields of cell biology and physiology, stem cell research, regenerative medicine, cancer research, drug discovery, and gene and protein expression studies. In addition,3D models can provide unique insight into bacteriology, virology, parasitology and host-pathogen interactions. This review summarizes and analyzes recent progress in human virological research with 3D cell culture models. We discuss viral growth, replication, proliferation, infection, virus-host interactions and antiviral drugs in 3D culture models.

Therapeutic strategies of three-dimensional stem cell spheroids and organoids for tissue repair and regeneration

Three-dimensional(3D)stem cell culture systems have attracted considerable attention as a way to better mimic the complex interactions between individual cells and the extracellular matrix(ECM)that occur in vivo.Moreover,3D cell culture systems have unique properties that help guide specific functions,growth,and processes of stem cells(e.g.,embryogenesis,morphogenesis,and organogenesis).Thus,3D stem cell culture systems that mimic in vivo environments enable basic research about various tissues and organs.In this review,we focus on the advanced therapeutic applications of stem cell-based 3D culture systems generated using different engineering techniques.Specifically,we summarize the historical advancements of 3D cell culture systems and discuss the therapeutic applications of stem cell-based spheroids and organoids,including engineering techniques for tissue repair and regeneration.

Application and Progress of Cultured Models of Gallbladder Carcinoma

Gallbladder carcinoma (GBC) is a malignant tumor of the bil-iary system that is aggressive, difficult to detect early, and has a low surgical resection rate and poor prognosis. Ap-propriate in vitro growth models are expected to focus on the study of the biological behavior and assess treatment effects. Nonetheless, cancer initiation, progression, and in-vasion include spatiotemporal changes and changes in the cell microenvironment intracellular communication, and in-tracellular molecules, making the development of in vitro growth models very challenging. Recent advances in bioma-terial methods and tissue engineering, particularly advances in bioprinting procedures, have paved the way for advances in the creative phase of in vitro cancer research. To date, an increasing number of cultured models of gallbladder disease have emerged, such as two-dimensional (2D) GBC growth cell cultures, three-dimensional (3D) GBC growth cell cul-tures, xenograft models, and 3D bioprinting methods. These models can serve as stronger platforms, focusing on tumor growth initiation, the association with the microenvironment, angiogenesis, motility, aggression, and infiltration. Bioprint-ed growth models can also be used for high-throughput drug screening and validation, as well as translational opportuni-ties for individual cancer therapy. This study focused on the exploration, progress, and significance of the development of GBC cultural models. We present our views on the short-comings of existing models, investigate new innovations, and plan future improvements and application possibilities for cancer models.

Bioactive polypeptide hydrogels modified with RGD and N-cadherin mimetic peptide promote chondrogenic differentiation of bone marrow mesenchymal stem cells

Cell-material and cell-cell interactions represent two crucial aspects of the regulation of cell behavior.In the present study,poly(L-glutamic acid)(PLG)hydrogels were prepared by catalyst-free click crosslinking via a strain-promoted azide-alkyne cycloaddition(SPAAC)reaction between azido-grafted PLG(PLG-N3)and azadibenzocyclooctyne-grafted PLG(PLG-ADIBO).The bioactive peptides c(RGDfK)and N-cadherin mimetic peptide(N-Cad)were both conjugated to the PLG hydrogel(denoted PLG+RGD/N-Cad)in order to regulate cell-material and cell-cell interactions.Gelation time and storage modulus of the hydrogels were tunable through variations in the concentration of polypeptide precursors.The hydrogels degraded gradually in the presence of proteinases.The viability of bone marrow mesenchymal stem cells(BMSCs)was maintained when cultured with extracts of the hydrogels or encapsulated within the hydrogels.Degradation was observed within 10 weeks following the subcutaneous injection of hydrogel solution in rats,displaying excellent histocompatibility in vivo.The introduction of RGD into the PLG hydrogel promoted the adhesion of BMSCs onto the hydrogels.Moreover,when encapsulated within the PLG+RGD/NCad hydrogel,BMSCs secreted cartilage-specific matrix,in addition to chondrogenic gene and protein expression being significantly enhanced in comparison with BMSCs encapsulated in hydrogels without N-Cad modification.These findings suggest that these biodegradable,bioactive polypeptide hydrogels have great potential for use in 3D cell culture and in cartilage tissue engineering.