Effect of Hydrogel Stiffness on Chemoresistance of Breast Cancer Cells in 3D Culture.

Chemotherapy is one of the most common strategies for cancer treatment, whereas drug resistance reduces the efficiency of chemotherapy and leads to treatment failure. The mechanism of emerging chemoresistance is complex and the effect of extracellular matrix (ECM) surrounding cells may contribute to drug resistance. Although it is well known that ECM plays an important role in orchestrating cell functions, it remains exclusive how ECM stiffness affects drug resistance. In this study, we prepared agarose hydrogels of different stiffnesses to investigate the effect of hydrogel stiffness on the chemoresistance of breast cancer cells to doxorubicin (DOX). Agarose hydrogels with a stiffness range of 1.5 kPa to 112.3 kPa were prepared and used to encapsulate breast cancer cells for a three-dimensional culture with different concentrations of DOX. The viability of the cells cultured in the hydrogels was dependent on both DOX concentration and hydrogel stiffness. Cell viability decreased with DOX concentration when the cells were cultured in the same stiffness hydrogels. When DOX concentration was the same, breast cancer cells showed higher viability in high-stiffness hydrogels than they did in low-stiffness hydrogels. Furthermore, the expression of P-glycoprotein mRNA in high-stiffness hydrogels was higher than that in low-stiffness hydrogels. The results suggested that hydrogel stiffness could affect the resistance of breast cancer cells to DOX by regulating the expression of chemoresistance-related genes.

Smart composite scaffold to synchronize magnetic hyperthermia and chemotherapy for efficient breast cancer therapy.

Combination of different therapies is an attractive approach for cancer therapy. However, it is a challenge to synchronize different therapies for maximization of therapeutic effects. In this work, a smart composite scaffold that could synchronize magnetic hyperthermia and chemotherapy was prepared by hybridization of magnetic Fe3O4 nanoparticles and doxorubicin (Dox)-loaded thermosensitive liposomes with biodegradable polymers. Irradiation of alternating magnetic field (AMF) could not only increase the scaffold temperature for magnetic hyperthermia but also trigger the release of Dox for chemotherapy. The two functions of magnetic hyperthermia and chemotherapy were synchronized by switching AMF on and off. The synergistic anticancer effects of the composite scaffold were confirmed by in vitro cell culture and in vivo animal experiments. The composite scaffold could efficiently eliminate breast cancer cells under AMF irradiation. Moreover, the scaffold could support proliferation and adipogenic differentiation of mesenchymal stem cells for adipose tissue reconstruction after anticancer treatment. In vivo regeneration experiments showed that the composite scaffolds could effectively maintain their structural integrity and facilitate the infiltration and proliferation of normal cells within the scaffolds. The composite scaffold possesses multi-functions and is attractive as a novel platform for efficient breast cancer therapy.

A neutralizable dimeric anti-thrombin aptamer with potent anticoagulant activity in mice.

Heparin-induced thrombocytopenia (HIT) is a complication caused by administration of the anticoagulant heparin. Although the number of patients with HIT has drastically increased because of coronavirus disease 2019 (COVID-19), the currently used thrombin inhibitors for HIT therapy do not have antidotes to arrest the severe bleeding that occurs as a side effect; therefore, establishment of safer treatments for HIT patients is imperative. Here, we devised a potent thrombin inhibitor based on bivalent aptamers with a higher safety profile via combination with the antidote. Using an anti-thrombin DNA aptamer M08s-1 as a promising anticoagulant, its homodimer and heterodimer with TBA29 linked by a conformationally flexible linker or a rigid duplex linker were designed. The dimerized M08s-1-based aptamers had about 100-fold increased binding affinity to human and mouse thrombin compared with the monomer counterparts. Administration of these bivalent aptamers into mice revealed that the anticoagulant activity of the dimers significantly surpassed that of an approved drug for HIT treatment, argatroban. Moreover, adding protamine sulfate as an antidote against the most potent bivalent aptamer completely suppressed the anticoagulant activity of the dimer. Emerging potent and neutralizable anticoagulant aptamers will be promising candidates for HIT treatment with a higher safety profile.

Locally Administered Photodynamic Therapy for Cancer Using Nano-Adhesive Photosensitizer.

Photodynamic therapy (PDT) is a great potential anti-tumor therapy owing to its non-invasiveness and high spatiotemporal selectivity. However, systemically administered photosensitizers diffuse in the skin and the eyes for a long duration, which cause phototoxicity to bright light and sunlight. Therefore, following PDT, patients must avoid exposure of to light and sunlight to avoid this phototoxicity. In this study, we have developed a locally administered PDT using nano-adhesive porphyrin with polycations consisting of quaternary ammonium salt groups (aHP) as a photosensitizer. The aHP, approximately 3.0 nm in diameter, adhered the negatively charged cell membrane via electrostatic interaction. The aHP localized to the endosome via cell adhesion and induced apoptosis upon 635 nm light irradiation. On being administered subcutaneously on the tumor, 30% of the injected aHP remained in the administered sites. However, low-molecular-weight hematoporphyrin dihydrochloride (HP) disappeared due to rapid diffusion. PDT with locally administered aHP showed a higher anti-tumor effect after light irradiation at 635 nm for three days compared to low-molecular-weight HP. Intraperitoneal administration of HP caused severe phototoxicity upon irradiation with ultraviolet A at 10 J cm-2, whereas aHP did not cause phototoxicity because its diffusion into the skin could be suppressed, probably due to the high-molecular weight of aHP. Therefore, locally administered PDT with aHP is a potential PDT having high therapeutic efficacy without phototoxicity.

3D culture of bovine articular chondrocytes in viscous medium encapsulated in agarose hydrogels for investigation of viscosity influence on cell functions.

The mechanical properties of an extracellular microenvironment can affect cell functions. The effects of elasticity and viscoelasticity on cell functions have been extensively studied with hydrogels of tunable mechanical properties. However, investigation of the viscosity effect on cell functions is still very limited and it can be tricky to explore how viscosity affects cells in three-dimensional (3D) culture due to the lack of appropriate tools. In this study, agarose hydrogel containers were prepared and used to encapsulate viscous media for 3D cell culture to investigate the viscosity effect on the functions of bovine articular chondrocytes (BACs). Polyethylene glycol of different molecular weights was used to adjust culture medium viscosity in a large range (72.8-679.2 mPa s). The viscosity affected gene expression and secretion of cartilagenious matrices, while it did not affect BAC proliferation. The BACs cultured in the lower viscosity medium (72.8 mPa s) showed a higher level of cartilaginous gene expression and matrix secretion.

Singlet oxygen-generating cell-adhesive glass surfaces for the fundamental investigation of plasma membrane-targeted photodynamic therapy.

Recently, plasma membrane-targeted photodynamic therapy has attracted attention as an effective cancer immunotherapeutic strategy. However, the released photosensitizers do not only adhere to the plasma membrane but may also be internalized in the cytosol, in endosomes/lysosomes, hindering investigations of the effects of photosensitizers attached to the plasma membrane. In this study, we developed a cell culture dish with singlet oxygen-generating cell-adhesive glass surfaces that allows investigation of the effects of photosensitizers attached to the plasma membrane. For cell adhesion, poly[N-(3-aminopropyl)methacrylamide] conjugated with hematoporphyrin PA-HpD was immobilized on the glass surfaces. Singlet oxygen was produced from the PA-HpD-immobilized glass surface upon laser irradiation at 635 nm. When murine colon adenocarcinoma 26 (Colon-26) cells were cultured on the PA-HpD-immobilized surface, the cells were swollen and ruptured, leading to effective apoptotic cell death using laser irradiation at 635 nm. In addition, microvesicles of approximately 10 µm in diameter were released from the plasma membrane into the culture medium. These phenomena were due to the oxidation of lipids in the cellular membrane, caused by the plasma membrane-targeted photodynamic therapy. In contrast, these phenomena were not observed on poly[N-(3-aminopropyl)methacrylamide]-immobilized glass surfaces. These results indicate that cell culture dishes with singlet oxygen-generating cell-adhesive glass surfaces can be used to investigate fundamental mechanisms in plasma membrane-targeted photodynamic therapy.

How Hydrogel Stiffness Affects Adipogenic Differentiation of Mesenchymal Stem Cells under Controlled Morphology.

Matrix stiffness has been disclosed as an essential regulator of cell fate. However, it is barely studied how the matrix stiffness affects stem cell functions when cell morphology changes. Thus, in this study, the effect of hydrogel stiffness on adipogenic differentiation of human bone-marrow-derived mesenchymal stem cells (hMSCs) with controlled morphology was investigated. Micropatterns of different size and elongation were prepared by a photolithographical micropatterning technique. The hMSCs were cultured on the micropatterns and showed a different spreading area and elongation following the geometry of the underlying micropatterns. The cells with controlled morphology were embedded in agarose hydrogels of different stiffnesses. The cells showed a different level of adipogenic differentiation that was dependent on both hydrogel stiffness and cell morphology. Adipogenic differentiation became strong when the cell spreading area decreased and hydrogel stiffness increased. Adipogenic differentiation did not change with cell elongation. Therefore, cell spreading area and hydrogel stiffness could synergistically affect adipogenic differentiation of hMSCs, while cell elongation did not affect adipogenic differentiation. A change of cell morphology and hydrogel stiffness was accompanied by actin filament alignment that was strongly related to adipogenic differentiation. The results indicated that cell morphology could affect cellular sensitivity to hydrogel stiffness. The results will provide useful information for the elucidation of the interaction of stem cells and their microenvironmental biomechanical cues.

Acyl plastoquinol is a major cyanobacterial substance that co-migrates with triacylglycerol in thin-layer chromatography.

Various studies have suggested the presence of triacylglycerol in cyanobacteria, but no convincing evidence exists. We purified a substance co-migrating with triacylglycerol in thin-layer chromatography and determined its structure using mass spectrometry, gas chromatography, and 1H and 13C NMR. The major components were palmitoyl and stearoyl plastoquinols (acyl plastoquinol). Acyl plastoquinol has never been described before, although acyloxy derivative of plastoquione has been described as plastoquinone B. The level of acyl plastoquinol was 0.4% of the total lipids. We still do not have clear evidence for the presence of triacylglycerol. If present, the maximum triacylglycerol level must be at most 10% of acyl plastoquinol. The Synechocystis Slr2103 protein was suggested to synthesize triacylglycerol, but the product could be acyl plastoquinol. The possible roles of this novel compound in photosynthesis should be a new focus of research.

Composite Scaffolds of Gelatin and Fe3 O4 Nanoparticles for Magnetic Hyperthermia-Based Breast Cancer Treatment and Adipose Tissue Regeneration.

Postsurgical treatment of breast cancer remains a challenge with regard to killing residual cancer cells and regenerating breast defects. To prepare composite scaffolds for postoperative use, gelatin is chemically modified with folic acid (FA) and used for hybridization with citrate-modified Fe3 O4 nanoparticles (Fe3 O4 -citrate NPs) to fabricate Fe3 O4 /gelatin composite scaffolds which pore structures are controlled by free ice microparticles. The composite scaffolds have large spherical pores that are interconnected to facilitate cell entry and exit. The FA-functionalized composite scaffolds have the ability to capture breast cancer cells. The Fe3 O4 /gelatin composite scaffolds possess a high capacity for magnetic-thermal conversion to ablate breast cancer cells during alternating magnetic field (AMF) irradiation. In addition, the composite scaffolds facilitate the growth and adipogenesis of mesenchymal stem cells. The composite scaffolds have multiple functions for eradication of residual cancer cells under AMF irradiation and for regeneration of resected adipose tissue when AMF is off.

Stepwise photothermal therapy and chemotherapy by composite scaffolds of gold nanoparticles, BP nanosheets and gelatin immobilized with doxorubicin-loaded thermosensitive liposomes.

In recent years, the synergistic effect of photothermal therapy (PTT) and chemotherapy has been recognized as an effective strategy for cancer treatment. Controlling the PTT temperature and drug release profile is desirable for minimizing the unexpected damage to normal cells. In this study, a smart platform of stepwise PTT and chemotherapy has been developed by using composite porous scaffolds of biodegradable black phosphorus (BP) nanosheets, gold nanorods(AuNRs), doxorubicin (Dox)-encapsulated thermosensitive liposomes and biodegradable polymers. Under near-infrared (NIR) laser irradiation, the composite scaffolds could attain high and low local temperatures before and after BP degradation, respectively. Dox release from the composite scaffolds could be controlled by the temperature change. In vitro cell culture and in vivo animal experiments indicated that a strong synergistic effect of PTT and chemotherapy could be achieved at an early stage of treatment before BP degradation, and a mild hyperthermia effect was shown for chemotherapy in the late stage after BP degradation. Moreover, the composite scaffolds after the complete release of Dox could support the proliferation of mesenchymal stem cells. The composite scaffolds showed a synergistic effect of stepwise PTT and chemotherapy for breast cancer elimination and promoted stem cell activities after killing cancer cells.

Preparation of composite scaffolds composed of gelatin and Au nanostar-deposited black phosphorus nanosheets for the photothermal ablation of cancer cells and adipogenic differentiation of stem cells.

Photothermal nanoparticles are important in photothermal therapy. Combining different nanoparticles can achieve a high photothermal capacity. In this study, composite nanoparticles composed of black phosphorus nanosheets (BPNSs) and gold nanostars (BP-AuNSs) were synthesized by using BPNSs as the reductant. AuNSs were deposited on the BPNSs. The BP-AuNSs were further hybridized with porous gelatin scaffolds to prepare gelatin-BP-AuNS composite scaffolds. The gelatin-BP-AuNS composite scaffolds promoted cell migration and distribution. The synergistic effects of the BPNSs and AuNSs endowed the gelatin-BP-AuNS composite scaffolds with excellent photothermal properties. The gelatin-BP-AuNS composite scaffolds eliminated cancer cells after near infrared laser exposure and supported the adipogenic differentiation of human mesenchymal stem cells. Thus, this gelatin-BP-AuNS composite scaffold holds promise for breast cancer therapy.

Morphological Dependence of Breast Cancer Cell Responses to Doxorubicin on Micropatterned Surfaces.

Cell morphology has been widely investigated for its influence on the functions of normal cells. However, the influence of cell morphology on cancer cell resistance to anti-cancer drugs remains unclear. In this study, micropatterned surfaces were prepared and used to control the spreading area and elongation of human breast cancer cell line. The influences of cell adhesion area and elongation on resistance to doxorubicin were investigated. The percentage of apoptotic breast cancer cells decreased with cell spreading area, while did not change with cell elongation. Large breast cancer cells had higher resistance to doxorubicin, better assembled actin filaments, higher DNA synthesis activity and higher expression of P-glycoprotein than small breast cancer cells. The results suggested that the morphology of breast cancer cells could affect their resistance to doxorubicin. The influence was correlated with cytoskeletal organization, DNA synthesis activity and P-glycoprotein expression.

Stepwise Proliferation and Chondrogenic Differentiation of Mesenchymal Stem Cells in Collagen Sponges under Different Microenvironments.

Biomimetic microenvironments are important for controlling stem cell functions. In this study, different microenvironmental conditions were investigated for the stepwise control of proliferation and chondrogenic differentiation of human bone-marrow-derived mesenchymal stem cells (hMSCs). The hMSCs were first cultured in collagen porous sponges and then embedded with or without collagen hydrogels for continual culture under different culture conditions. The different influences of collagen sponges, collagen hydrogels, and induction factors were investigated. The collagen sponges were beneficial for cell proliferation. The collagen sponges also promoted chondrogenic differentiation during culture in chondrogenic medium, which was superior to the effect of collagen sponges embedded with hydrogels without loading of induction factors. However, collagen sponges embedded with collagen hydrogels and loaded with induction factors had the same level of promotive effect on chondrogenic differentiation as collagen sponges during in vitro culture in chondrogenic medium and showed the highest promotive effect during in vivo subcutaneous implantation. The combination of collagen sponges with collagen hydrogels and induction factors could provide a platform for cell proliferation at an early stage and subsequent chondrogenic differentiation at a late stage. The results provide useful information for the chondrogenic differentiation of stem cells and cartilage tissue engineering.

Long-Term Fluorescent Tissue Marking Using Tissue-Adhesive Porphyrin with Polycations Consisting of Quaternary Ammonium Salt Groups.

Localization of tumors during laparoscopic surgery is generally performed by locally injecting India ink into the submucosal layer of the gastrointestinal tract using endoscopy. However, the location of the tumor is obscured because of the black-stained surgical field and the blurring caused by India ink. To solve this problem, in this study, we developed a tissue-adhesive porphyrin with polycations consisting of quaternary ammonium salt groups. To evaluate the ability of tissue-adhesive porphyrin in vivo, low-molecular-weight hematoporphyrin and tissue-adhesive porphyrin were injected into the anterior wall of the exposed stomach in rats. Local injection of low-molecular-weight hematoporphyrin into the anterior wall of the stomach was not visible even after 1 day because of its rapid diffusion. In contrast, the red fluorescence of the tissue-adhesive porphyrin was visible even after 7 days due to the electrostatic interactions between the positively-charged moieties of the polycation in the tissue-adhesive porphyrin and the negatively-charged molecules in the tissue. In addition, intraperitoneal injection of tissue-adhesive porphyrin in rats did not cause adverse effects such as weight loss, hepatic or renal dysfunction, or organ adhesion in the abdominal cavity. These results indicate that tissue-adhesive porphyrin is a promising fluorescent tissue-marking agent.

Three-dimensional culture and chondrogenic differentiation of mesenchymal stem cells in interconnected collagen scaffolds.

Interconnected scaffolds are useful for promoting the chondrogenic differentiation of stem cells. Collagen scaffolds with interconnected pore structures were fabricated with poly(lactic acid-co-glycolic acid) (PLGA) sponge templates. The PLGA-templated collagen scaffolds were used to culture human bone marrow-derived mesenchymal stem cells (hMSCs) to investigate their promotive effect on the chondrogenic differentiation of hMSCs. The cells adhered to the scaffolds with a homogeneous distribution and proliferated with culture time. The expression of chondrogenesis-related genes was upregulated, and abundant cartilaginous matrices were detected. After subcutaneous implantation, the PLGA-templated collagen scaffolds further enhanced the production of cartilaginous matrices and the mechanical properties of the implants. The good interconnectivity of the PLGA-templated collagen scaffolds promoted chondrogenic differentiation. In particular, the collagen scaffolds prepared with large pore-bearing PLGA sponge templates showed the highest promotive effect.

Near-Infrared Light Irradiation of Porphyrin-Modified Gold Nanoparticles Promotes Cancer-Cell-Specific Cytotoxicity.

The use of nanoparticles has been investigated as a new cancer treatment. These can induce specific cytotoxicity in cancer cells. In particular, Au nanoparticles (AuNPs) have unique characteristics. The maximum absorption spectrum of AuNPs can be adjusted to modify their size or shape to absorb near-infrared light that can penetrate into tissue without photodamage. Thus, the combination of AuNPs and near-infrared light can be used to treat cancer in deep-seated organs. To obtain effective cancer-specific accumulation of AuNPs, we focused on porphyrin and synthesized a porphyrin-attached Au compound: Au-HpD. In this study, we investigated whether Au-HpD possesses cancer-specific accumulation and cytotoxicity. Intracellular Au-HpD accumulation was higher in cancer cells than in normal cells. In order to analyze the cytotoxicity induced by Au-HpD, cancer cells and normal cells were co-cultured in the presence of Au-HpD; then, they were subjected to 870 nm laser irradiation. We observed that, after laser irradiation, cancer cells showed significant morphological changes, such as chromatin condensation and nuclear fragmentation indicative of cell apoptosis. This strong effect was not observed when normal cells were irradiated. Moreover, cancer cells underwent cell apoptosis with combination therapy.

Self-Assembling Antioxidants for Ischemia-Reperfusion Injuries.

Significance: Ischemia-reperfusion (IR) injury is a major component of severe damage in vascular occlusion during stroke, myocardial infarction, surgery, and organ transplantation, and is exacerbated by the excessive generation of reactive oxygen species (ROS), which occurs particularly during reperfusion. With the aging of the population, IR injury is becoming a serious problem in various organs, such as the kidney, brain, and heart, as well as in the mesenteric capillaries. Recent Advances: To prevent reperfusion injuries, natural and synthetic low-molecular-weight (LMW) antioxidants have been well studied. Critical Issues: However, these LMW antioxidants have various problems, including adverse effects due to excessive cellular uptake and their rapid clearance by the kidney, and cannot fully exert their potent antioxidant capacity in vivo. Future Directions: To overcome these problems, we designed and developed redox polymers with antioxidants covalently conjugated with them. These polymers self-assemble into nanoparticles in aqueous media, referred to as redox nanoparticles (RNPs). RNPs suppress their uptake into normal cells, accumulate at inflammation sites, and effectively scavenge ROS in damaged tissues. We had developed two types of RNPs: RNPN, which disintegrates in response to acidic pH; and RNPO, which does not collapse, regardless of the environmental pH. Utilizing the pH-sensitive and -insensitive characteristics of RNPN and RNPO, respectively, RNPs were found to exhibit remarkable therapeutic effects on various oxidative stress disorders, including IR injuries. Thus, RNPs are promising nanomedicines for use as next-generation antioxidants. This review summarizes the therapeutic impacts of RNPs in the treatment of kidney, cerebral, myocardial, and intestinal IR injuries. Antioxid. Redox Signal. 36, 70-80.

PLGA-collagen-BPNS Bifunctional composite mesh for photothermal therapy of melanoma and skin tissue engineering.

The treatment of melanoma requires not only the elimination of skin cancer cells but also skin regeneration to heal defects. To achieve this goal, a bifunctional composite scaffold of poly(DL-lactic-co-glycolic acid) (PLGA), collagen and black phosphorus nanosheets (BPNSs) was prepared by hybridizing a BPNS-embedded collagen sponge with a PLGA knitted mesh. The composite mesh increased the temperature under near-infrared laser irradiation. The incorporation of BPNSs provided the PLGA-collagen-BPNS composite mesh with excellent photothermal properties for the photothermal ablation of melanoma cells both in vitro and in vivo. The PLGA-collagen-BPNS composite mesh had high mechanical strength for easy handling. The PLGA-collagen-BPNS composite mesh facilitated the proliferation of fibroblasts, promoted the expression of angiogenesis-related genes and the genes of components of the extracellular matrix for skin tissue regeneration. The high mechanical strength, photothermal ablation capability and skin tissue regeneration effects demonstrate that the bifunctional PLGA-collagen-BPNS composite mesh is a versatile and effective platform for the treatment of melanoma and the regeneration of skin defects.

Reduced cytotoxicity of polyethyleneimine by covalent modification of antioxidant and its application to microalgal transformation.

The conversion of carbon dioxide into valuable chemicals is an effective strategy for combating augmented concentrations of carbon dioxide in the environment. Microalgae photosynthetically produce valuable chemicals that are used as biofuels, sources for industrial materials, medicinal leads, and food additives. Thus, improvements in microalgal technology via genetic engineering may prove to be promising for the tailored production of novel metabolites. For the transformation of microalgae, nucleic acids such as plasmid DNA (pDNA) are delivered into the cells using physical and mechanical techniques, such as electroporation, bombardment with DNA-coated microprojectiles, and vortexing with glass beads. However, owing to the electrostatic repulsion between negatively charged cell walls and nucleic acids, the delivery of nucleic acids into the microalgal cells is challenging. To solve this issue, in this study, we investigated microalgal transformation via electroporation using polyplexes with linear polyethyleneimine (LPEI) and pDNA. However, the high toxicity of LPEI decreased the transformation efficiency in Chlamydomonas reinhardtii cells. We revealed that the toxicity of LPEI was due to oxidative stress resulting from the cellular uptake of LPEI. To suppress the toxicity of LPEI, an antioxidant, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), was covalently conjugated with LPEI; the conjugate was named as TEMPO-LPEI. Interestingly, with a cellular uptake tendency similar to that of LPEI, TEMPO-LPEI dramatically decreased oxidative stress and cytotoxicity. Electroporation using polyplexes of TEMPO-LPEI and pDNA enhanced the transformation efficiency, compared to those treated with bare pDNA and polyplexes of LPEI/pDNA. This result indicates that polycations conjugated with antioxidants could be useful in facilitating microalgal transformation.

Interconnected collagen porous scaffolds prepared with sacrificial PLGA sponge templates for cartilage tissue engineering.

Interconnected pore structures of scaffolds are important to control the cell functions for cartilage tissue engineering. In this study, collagen scaffolds with interconnected pore structures were prepared using poly(D,L-lactide-co-glycolide) (PLGA) sponges as sacrificial templates. Six types of PLGA sponges of different pore sizes and porosities were prepared by the solvent casting/particulate leaching method and used to regulate the interconnectivity of the collagen scaffolds. The integral and continuous templating structure of PLGA sponges generated well-interconnected pore structures in the collagen scaffolds. Bovine articular chondrocytes cultured in collagen scaffolds showed homogenous distribution, fast proliferation, high expression of cartilaginous genes and high secretion of cartilaginous extracellular matrix. In particular, the collagen scaffold templated by the PLGA sacrificial sponge that was prepared with a high weight ratio of PLGA and large salt particulates showed the most promotive effect on cartilage tissue formation. The interconnected pore structure facilitated cell distribution, cell-cell interaction and cartilage tissue regeneration.