A mechanical composite spheres analysis of engineered cartilage dynamics

In the preparation of bioengineered reparative strategies for damaged or diseased tissues, the processes of biomaterial degradation and neotissue synthesis combine to affect the developing mechanical state of multiphase, composite engineered tissues. Here, cell-polymer constructs for engineered cartilage have been fabricated by seeding chondrocytes within three-dimensional scaffolds of biodegradable polymers. During culture, synthetic scaffolds degraded passively as the cells assembled an extracellular matrix (ECM) composed primarily of glycosaminoglycan and collagen. Biochemical and biomechanical assessment of the composite (cells, ECM, and polymer scaffold) were modeled at a unit-cell level to mathematically solve stress-strain relationships and thus construct elastic properties (n=4 samples per seven time points). This approach employed a composite spheres, micromechanical analysis to determine bulk moduli of: (1) the cellular-ECM inclusion within the supporting scaffold structure; and (2) the cellular inclusion within its ECM. Results indicate a dependence of constituent volume fractions with culture time (p<0.05). Overall mean bulk moduli were variably influenced by culture, as noted for the cell-ECM inclusion (K(c-m)=29.7 kPa, p=0.1439), the cellular inclusion (K(c)=5.5 kPa, p=0.0067), and its surrounding ECM (K(m)=373.9 kPa, p=0.0748), as well as the overall engineered construct (K=165.0 kPa, p=0.6899). This analytical technique provides a framework to describe the time-dependent contribution of cells, accumulating ECM, and a degrading scaffold affecting bioengineered construct mechanical properties.     

Reprogramming cardiomyocyte mechanosensing by crosstalk between integrins and hyaluronic acid receptors

The elastic modulus of bioengineered materials has a strong influence on the phenotype of many cells including cardiomyocytes. On polyacrylamide (PAA) gels that are laminated with ligands for integrins, cardiac myocytes develop well organized sarcomeres only when cultured on substrates with elastic moduli in the range 10 kPa-30 kPa, near those of the healthy tissue. On stiffer substrates (>60 kPa) approximating the damaged heart, myocytes form stress fiber-like filament bundles but lack organized sarcomeres or an elongated shape. On soft (<1 kPa) PAA gels myocytes exhibit disorganized actin networks and sarcomeres. However, when the polyacrylamide matrix is replaced by hyaluronic acid (HA) as the gel network to which integrin ligands are attached, robust development of functional neonatal rat ventricular myocytes occurs on gels with elastic moduli of 200 Pa, a stiffness far below that of the neonatal heart and on which myocytes would be amorphous and dysfunctional when cultured on polyacrylamide-based gels. The HA matrix by itself is not adhesive for myocytes, and the myocyte phenotype depends on the type of integrin ligand that is incorporated within the HA gel, with fibronectin, gelatin, or fibrinogen being more effective than collagen I. These results show that HA alters the integrin-dependent stiffness response of cells in vitro and suggests that expression of HA within the extracellular matrix (ECM) in vivo might similarly alter the response of cells that bind the ECM through integrins. The integration of HA with integrin-specific ECM signaling proteins provides a rationale for engineering a new class of soft hybrid hydrogels that can be used in therapeutic strategies to reverse the remodeling of the injured myocardium.     

Gelatin interpenetration in poly N-isopropylacrylamide network reduces the compressive modulus of the scaffold: A property employed to mimic hepatic matrix stiffness

The progression of liver disease from normal to cirrhotic state is characterized by modulation of the stiffness of the extracellular matrix (ECM). Mimicking this modulation in vitro scaffold could provide a better insight into hepatic cell behavior. In this study, interpenetrating poly(N-isopropylacrylamide-co-gelatin) cryogels were synthesized in 48 different compositions to yield scaffolds of different properties. It was observed that a high concentration of N-isopropylacrylamide (NIPAAm) leads to the formation of small pores while gelatin interpenetration on poly-NIPAAm framework renders porous structure. Swelling properties and porosity of the gels decreased with an increase in NIPAAm concentration owing to the increased compactness of the gels. Gelatin interpenetration relaxed the gels and enhanced these properties. An increase in gelatin concentration led to a reduction in compressive moduli indicating that gelatin interpenetration in the poly-NIPAAm network softens the cryogel. With the increase in NIPAAm concentration, the effect of gelatin interpenetration in reducing the compressive moduli expanded. The cytocompatibility studies indicated that the gels are cell-adherent and compatible with HepG2. Furthermore, biochemical and real-time polymerase chain reaction studies revealed that HepG2 and Huh-7 cells cultured on scaffolds mimicking the ECM stiffness of normal liver (1.5–2.5 kPa) exhibited optimum liver-specific functionalities. Increasing the stiffness to fibrotic (4–9 kPa) and cirrhotic (10–20 kPa) ECM decreases the functionality.     

Fabrication of gelatin-hyaluronic acid hybrid scaffolds with tunable porous structures for soft tissue engineering

The development of three-dimensional (3-D) scaffolds with highly open porous structure is one of the most important issues in tissue engineering. In this study, 3-D macroporous gelatin/hyaluronic acid (GE/HA) hybrid scaffolds with varying porous morphology were prepared by freeze-drying their blending solutions and subsequent chemical crosslinking by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). The resulting scaffolds were characterized by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). Their swelling, in vitro degradation properties and compressive strength were also investigated. To evaluate in vitro cytocompatibility of scaffolds, mouse L929 fibroblasts were seeded onto the scaffolds for cell morphology and cell viability studies. It was found that the porous structure of scaffolds can be tailored by varying the ratios of gelatin to HA, both the swelling ratios and degradation rate increased with the increase of HA content in hybrid scaffolds, and crosslinking the scaffolds with EDC improved the degradation resistance of the scaffold in culture media and increased the mechanical strength of scaffolds. The in vitro results revealed that the prepared scaffolds do not induce cytotoxic effects and suitable for cell growth, especially in the case of scaffolds with higher gelatin content. The combined results of the physicochemical and biological studies suggested that the developed GE/HA hybrid scaffolds exhibit good potential and biocompatibility for soft tissue engineering applications.     

The influence of fibrous elastomer structure and porosity on matrix organization

Fibrous scaffolds are finding wide use in the field of tissue engineering, as they can be designed to mimic many native tissue properties and structures (e.g., cardiac tissue, meniscus). The influence of fiber alignment and scaffold architecture on cellular interactions and matrix organization was the focus of this study. Three scaffolds were fabricated from the photocrosslinkable elastomer poly(glycerol sebacate) (PGS), with changes in fiber alignment (non-aligned (NA) versus aligned (AL)) and the introduction of a PEO sacrificial polymer population to the AL scaffold (composite (CO)). PEO removal led to an increase in scaffold porosity and maintenance of scaffold anisotropy, as evident through visualization, mechanical testing, and mass loss studies. Hydrated scaffolds possessed moduli that ranged between ～3-240 kPa, failing within the range of properties (<300 kPa) appropriate for soft tissue engineering. CO scaffolds were completely degraded as early as 16 days, whereas NA and AL scaffolds had ～90% mass loss after 21 days when monitored in vitro. Neonatal cardiomyocytes, used as a representative cell type, that were seeded onto the scaffolds maintained their viability and aligned along the surface of the AL and CO fibers. When implanted subcutaneously in rats, a model that is commonly used to investigate in vivo tissue responses to biomaterials, CO scaffolds were completely integrated at 2 weeks, whereas ～13% and ～16% of the NA and AL scaffolds, respectively remained acellular. However, all scaffolds were completely populated with cells at 4 weeks post-implantation. Polarized light microscopy was used to evaluate the collagen elaboration and orientation within the scaffold. An increase in the amount of collagen was observed for CO scaffolds and enhanced alignment of the nascent collagen was observed for AL and CO scaffolds compared to NA scaffolds. Thus, these results indicate that the scaffold architecture and porosity are important considerations in controlling tissue formation.     

Formation of collagen-glycosaminoglycan blended nanofibrous scaffolds and their biological properties

The development of blended collagen and glycosaminoglycan (GAG) scaffolds can potentially be used in many soft tissue engineering applications since the scaffolds mimic the structure and biological function of native extracellular matrix (ECM). In this study, we were able to obtain novel nanofibrous collagen-GAG scaffolds by electrospinning collagen blended with chondroitin sulfate (CS), a widely used GAG, in a mixed solvent of trifluoroethanol and water. The electrospun collagen-GAG scaffold with 4% CS (COLL-CS-04) exhibited a uniform fiber structure with nanoscale diameters. A second collagen-GAG scaffold with 10% CS consisted of smaller diameter fibers but exhibited a broader diameter distribution due to the different solution properties in comparison with COLL-CS-04. After cross-linking with glutaraldehyde vapor, the collagen-GAG scaffolds became more biostable and were resistant to collagenase degradation. This is evidently a more favorable environment allowing increased proliferation of rabbit conjunctiva fibroblast on the scaffolds. Incorporation of CS into collagen nanofibers without cross-linking did not increase the biostability but still promoted cell growth. The potential of applying the nanoscale collagen-GAG scaffold in tissue engineering is significant since the nanodimension fibers made of natural ECM mimic closely the native ECM found in the human body. The high surface area characteristic of this scaffold may maximize cell-ECM interaction and promote tissue regeneration faster than other conventional scaffolds.     

Engineered Smooth Muscle Tissues: Regulating Cell Phenotype with the Scaffold

Culturing cells on three-dimensional, biodegradable scaffolds may create tissues suitable either for reconstructive surgery applications or as novel in vitro model systems. In this study, we have tested the hypothesis that the phenotype of smooth muscle cells (SMCs) in three-dimensional, engineered tissues is regulated by the chemistry of the scaffold material. Specifically, we have directly compared cell growth and patterns of extracellular matrix (ECM) (e.g. , elastin and collagen) gene expression on two types of synthetic polymer scaffolds and type I collagen scaffolds. The growth rates of SMCs on the synthetic polymer scaffolds were significantly higher than on type I collagen sponges. The rate of elastin production by SMCs on polyglycolic acid (PGA) scaffolds was 3.5 +/- 1.1-fold higher than that on type I collagen sponges on Day 11 of culture. In contrast, the collagen production rate on type I collagen sponges was 3.3 +/- 1.1-fold higher than that on PGA scaffolds. This scaffold-dependent switching between elastin and collagen gene expression was confirmed by Northern blot analysis. The finding that the scaffold chemistry regulates the phenotype of SMCs independent of the scaffold physical form was confirmed by culturing SMCs on two-dimensional films of the scaffold materials. It is likely that cells adhere to these scaffolds via different ligands, as the major protein adsorbed from the serum onto synthetic polymers was vitronectin, whereas fibronectin and vitronectin were present at high density on type I collagen sponges. In summary, this study demonstrates that three-dimensional smooth muscle-like tissues can be created by culturing SMCs on three-dimensional scaffolds, and that the phenotype of the SMCs is strongly regulated by the scaffold chemistry. These engineered tissues provide novel, three-dimensional models to study cellular interaction with ECM in vitro.     

Determination of mechanical properties of soft tissue scaffolds by atomic force microscopy nanoindentation

While the determination of mechanical properties of a hard scaffold is relatively straightforward, the mechanical testing of a soft tissue scaffold poses significant challenges due in part to its fragility. Here, we report a new approach for characterizing the stiffness and elastic modulus of a soft scaffold through atomic force microscopy (AFM) nanoindentation. Using collagen-chitosan hydrogel scaffolds as model soft tissue scaffolds, we demonstrated the feasibility of using AFM nanoindentation to determine a force curve of a soft tissue scaffold. A mathematical model was developed to ascertain the stiffness and elastic modulus of a scaffold from its force curve obtained under different conditions. The elastic modulus of a collagen-chitosan (80%/20%, v/v) scaffold is found to be 3.69 kPa. The scaffold becomes stiffer if it contains more chitosan. The elastic modulus of a scaffold composed of 70% collagen and 30% chitosan is about 11.6 kPa. Furthermore, the stiffness of the scaffold is found to be altered significantly by extracellular matrix deposited from cells that are grown inside the scaffold. The elastic modulus of collagen-chitosan scaffolds increased from 10.5 kPa on day 3 to 63.4 kPa on day 10 when human foreskin fibroblast cells grew inside the scaffolds. Data acquired from these measurements will offer new insights into understanding cell fate regulation induced by physiochemical cues of tissue scaffolds.     

Osteoid-mimicking dense collagen/chitosan hybrid gels

Bone extracellular matrix (ECM) is a 3D network, composed of collagen type I and a number of other macromolecules, including glycosaminoglycans (GAGs), which stimulate signaling pathways that regulate osteoblast growth and differentiation. To model the ECM of bone for tissue regenerative approaches, dense collagen/chitosan (Coll/CTS) hybrid hydrogels were developed using different proportions of CTS to mimic GAG components of the ECM. MC3T3-E1 mouse calvaria preosteoblasts were seeded within plastically compressed Coll/CTS hydrogels with solid content approaching that of native bone osteoid. Dense, cellular Coll/CTS hybrids were maintained for up to 8 weeks under either basal or osteogenic conditions. Higher CTS content significantly increased gel resistance to collagenase degradation. The incorporation of CTS to collagen gels decreased the apparent tensile modulus from 1.82 to 0.33 MPa. In contrast, the compressive modulus of Coll/CTS hybrids increased in direct proportion to CTS content exhibiting an increase from 23.50 to 55.25 kPa. CTS incorporation also led to an increase in scaffold resistance to cell-induced contraction. MC3T3-E1 viability, proliferation, and matrix remodeling capability (via matrix metalloproteinase expression) were maintained. Alkaline phosphatase activity was increased up to two-fold, and quantification of phosphate mineral deposition was significantly increased with CTS incorporation. Thus, dense Coll/CTS scaffolds provide osteoid-like models for the study of osteoblast differentiation and bone tissue engineering.     

Mesenchymal stem cell responses to bone-mimetic electrospun matrices composed of polycaprolactone, collagen I and nanoparticulate hydroxyapatite

The performance of biomaterials designed for bone repair depends, in part, on the ability of the material to support the adhesion and survival of mesenchymal stem cells (MSCs). In this study, a nanofibrous bone-mimicking scaffold was electrospun from a mixture of polycaprolactone (PCL), collagen I, and hydroxyapatite (HA) nanoparticles with a dry weight ratio of 50/30/20 respectively (PCL/col/HA). The cytocompatibility of this tri-component scaffold was compared with three other scaffold formulations: 100% PCL (PCL), 100% collagen I (col), and a bi-component scaffold containing 80% PCL/20% HA (PCL/HA). Scanning electron microscopy, fluorescent live cell imaging, and MTS assays showed that MSCs adhered to the PCL, PCL/HA and PCL/col/HA scaffolds, however more rapid cell spreading and significantly greater cell proliferation was observed for MSCs on the tri-component bone-mimetic scaffolds. In contrast, the col scaffolds did not support cell spreading or survival, possibly due to the low tensile modulus of this material. PCL/col/HA scaffolds adsorbed a substantially greater quantity of the adhesive proteins, fibronectin and vitronectin, than PCL or PCL/HA following in vitro exposure to serum, or placement into rat tibiae, which may have contributed to the favorable cell responses to the tri-component substrates. In addition, cells seeded onto PCL/col/HA scaffolds showed markedly increased levels of phosphorylated FAK, a marker of integrin activation and a signaling molecule known to be important for directing cell survival and osteoblastic differentiation. Collectively these results suggest that electrospun bone-mimetic matrices serve as promising degradable substrates for bone regenerative applications.     

Fabrication of porous polycaprolactone/hydroxyapatite (PCL/HA) blend scaffolds using a 3D plotting system for bone tissue engineering

For tissue engineering and regeneration, a porous scaffold with interconnected networks is needed to guide cell attachment and growth/ingrowth in three-dimensional (3D) structure. Using a rapid prototyping (RP) technique, we designed and fabricated 3D plotting system and three types of scaffolds: those from polycaprolactone (PCL), those from PCL and hydroxyapatite (HA), and those from PCL/HA and with a shifted pattern structure (PCL/HA/SP scaffold). Shifted pattern structure was fabricated to increase the cell attachment/adhesion. The PCL/HA/SP scaffold had a lower compressive modulus than PCL and PCL/HA scaffold. However, it has a better cell attachment than the scaffolds without a shifted pattern. MTT assay and alkaline phosphatase activity results for the PCL/HA/SP scaffolds were significantly enhanced compared to the results for the PCL and PCL/HA scaffolds. According to their degree of cell proliferation/differentiation, the scaffolds were in the following order: PCL/HA/SP > PCL/HA > PCL. These 3D scaffolds will be applicable for tissue engineering based on unique plotting system.     

Bioactive nanoparticles stimulate bone tissue formation in bioprinted three‐dimensional scaffold and human mesenchymal stem cells

Bioprinting based on thermal inkjet printing is a promising but unexplored approach in bone tissue engineering. Appropriate cell types and suitable biomaterial scaffolds are two critical factors to generate successful bioprinted tissue. This study was undertaken in order to evaluate bioactive ceramic nanoparticles in stimulating osteogenesis of printed bone marrow‐derived human mesenchymal stem cells (hMSCs) in poly(ethylene glycol)dimethacrylate (PEGDMA) scaffold. hMSCs suspended in PEGDMA were co‐printed with nanoparticles of bioactive glass (BG) and hydroxyapatite (HA) under simultaneous polymerization so the printed substrates were delivered with highly accurate placement in three‐dimensional (3D) locations. hMSCs interacted with HA showed the highest cell viability (86.62 ± 6.02%) and increased compressive modulus (358.91 ± 48.05 kPa) after 21 days in culture among all groups. Biochemical analysis showed the most collagen production and highest alkaline phosphatase activity in PEG‐HA group, which is consistent with gene expression determined by quantitative PCR. Masson's trichrome staining also showed the most collagen deposition in PEG‐HA scaffold. Therefore, HA is more effective comparing to BG for hMSCs osteogenesis in bioprinted bone constructs. Combining with our previous experience in vasculature, cartilage, and muscle bioprinting, this technology demonstrates the capacity for both soft and hard tissue engineering with biomimetic structures.     

Fabrication and evaluation of biomimetic-synthetic nanofibrous composites for soft tissue regeneration

Electrospun scaffolds hold promise for the regeneration of dense connective tissues, given their nanoscale topographies, provision of directional cues for infiltrating cells and versatile composition. Synthetic slow-degrading scaffolds provide long-term mechanical support and nanoscale instructional cues; however, these scaffolds suffer from a poor infiltration rate. Alternatively, nanofibrous constructs formed from natural biomimetic materials (such as collagen) rapidly infiltrate but provide little mechanical support. To take advantage of the positive features of these constructs, we have developed a composite scaffold consisting in both a biomimetic fiber fraction (i.e., Type I collagen nanofibers) together with a traditional synthetic (i.e., poly-[ε-caprolactone], PCL) fiber fraction. We hypothesize that inclusion of biomimetic elements will improve initial cell adhesion and eventual scaffold infiltration, whereas the synthetic elements will provide controlled and long-term mechanical support. We have developed a method of forming and crosslinking collagen nanofibers by using the natural crosslinking agent genipin (GP). Further, we have formed composites from collagen and PCL and evaluated the long-term performance of these scaffolds when seeded with mesenchymal stem cells. Our results demonstrate that GP crosslinking is cytocompatible and generates stable nanofibrous type I collagen constructs. Composites with varying fractions of the biomimetic and synthetic fiber families are formed and retain their collagen fiber fractions during in vitro culture. However, at the maximum collagen fiber fractions (20%), cell ingress is limited compared with pure PCL scaffolds. These results provide a new foundation for the development and optimization of biomimetic/synthetic nanofibrous composites for in vivo tissue engineering.     

Biodendrimer-based hydrogel scaffolds for cartilage tissue repair

Photo-crosslinkable dendritic macromolecules are attractive materials for the preparation of cartilage tissue engineering scaffolds that may be optimized for in situ formation of hydrated, mechanically stable, and well-integrated hydrogel scaffolds supporting chondrocytes and chondrogenesis. We designed and synthesized a novel hydrogel scaffold for cartilage repair, based on a multivalent and water-soluble tri-block copolymer consisting of a poly(ethylene glycol) core and methacrylated poly(glycerol succinic acid) dendrimer terminal blocks. The terminal methacrylates allow mild and biocompatible photo-crosslinking with a visible light, facilitating in vivo filling of irregularly shaped defects with the dendrimer-based scaffold. The multivalent dendrimer constituents allow high crosslink densities that inhibit swelling after crosslinking while simultaneously introducing biodegradation sites. The mechanical properties and water content of the hydrogel can easily be tuned by changing the biodendrimer concentration. In vitro chondrocyte encapsulation studies demonstrate significant synthesis of neocartilaginous material, containing proteoglycans and type II collagen.     

A cell leakproof PLGA‐collagen hybrid scaffold for cartilage tissue engineering

A cell leakproof porous poly(DL ‐lactic‐co‐glycolic acid) (PLGA)‐collagen hybrid scaffold was prepared by wrapping the surfaces of a collagen sponge except the top surface for cell seeding with a bi‐layered PLGA mesh. The PLGA‐collagen hybrid scaffold had a structure consisting of a central collagen sponge formed inside a bi‐layered PLGA mesh cup. The hybrid scaffold showed high mechanical strength. The cell seeding efficiency was 90.0% when human mesenchymal stem cells (MSCs) were seeded in the hybrid scaffold. The central collagen sponge provided enough space for cell loading and supported cell adhesion, while the bi‐layered PLGA mesh cup protected against cell leakage and provided high mechanical strength for the collagen sponge to maintain its shape during cell culture. The MSCs in the hybrid scaffolds showed round cell morphology after 4 weeks culture in chondrogenic induction medium. Immunostaining demonstrated that type II collagen and cartilaginous proteoglycan were detected in the extracellular matrices. Gene expression analyses by real‐time PCR showed that the genes encoding type II collagen, aggrecan, and SOX9 were upregulated. These results indicated that the MSCs differentiated and formed cartilage‐like tissue when being cultured in the cell leakproof PLGA‐collagen hybrid scaffold. The cell leakproof PLGA‐collagen hybrid scaffolds should be useful for applications in cartilage tissue engineering. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

The role of hydrogel structure and dynamic loading on chondrocyte gene expression and matrix formation

Crosslinked poly(ethylene glycol) (PEG) hydrogels are attractive scaffolds for cartilage tissue engineering because of their ability to mimic the aqueous environment and mechanical properties of native cartilage. In this study, hydrogel crosslinking density was varied to study the influence of gel structure and the application of dynamic loading (continuous, 1 Hz, 15% amplitude strain) on chondrocyte gene expression over 1 week culture. Gene expression was quantified using real-time RT-PCR for collagen II and aggrecan, the major cartilage extracellular matrix (ECM) components, and collagen I, an indicator of chondrocyte de-differentiation. When chondrocytes were encapsulated in PEG gels with low or high crosslinking, a high collagen II expression compared to collagen I expression (1000 or 100,000:1, respectively) indicated the native chondrocyte phenotype was retained. In the absence of loading, relative gene expression for collagen II and aggrecan was significantly higher (e.g., 2-fold and 4-fold, respectively, day 7) in the low crosslinked gels compared to gels with higher crosslinking. Dynamic loading, however, showed little effect on ECM gene expression in both crosslinked systems. To better understand the cellular environment, ECM production was qualitatively assessed using an in situ immunofluorescent technique and standard histology. A pericellular matrix (PCM) was observed as early as day 3 post-encapsulation and the degree of formation was dependent on gel crosslinking. These results suggest the PCM may protect the cells from sensing the applied loads. This study demonstrates that gel structure has a profound effect on chondrocyte gene expression, while dynamic loading has much less of an effect at early culture times.     

Encapsulating chondrocytes in degrading PEG hydrogels with high modulus: engineering gel structural changes to facilitate cartilaginous tissue production

A major challenge when designing cell scaffolds for chondrocyte delivery in vivo is creating scaffolds with sufficient mechanical properties to restore initial function while simultaneously controlling temporal changes in the gel structure to facilitate tissue formation. To address this design challenge, degradable photocrosslinked hydrogels based on poly(ethylene glycol) were investigated. To alter the gel's initial mechanical properties, hydrogels were fabricated by varying the initial macromer concentration from 10% to 15% to 20%. A twofold increase in macromer concentration resulted in an eightfold increase in the initial compressive modulus from 60 to 500 kPa. Gel degradation was tailored by incorporating fast-degrading crosslinks that enable maximal extracellular matrix (ECM) diffusion with time and a minimal number of nondegrading (or slowly degrading) crosslinks to maintain scaffold integrity and prevent complete gel erosion during tissue formation. Chondrocytes encapsulated in these gels produced cartilaginous tissue rich in glycosaminoglycans and collagen as seen biochemically and histologically. Interestingly, mass loss appeared to more closely match tissue secretion in gels fabricated from a 15% macromer concentration. However, the spatial ECM distribution was grossly similar in all three gels. By tailoring gel degradation and controlling network evolution during degradation, gels with optimal properties can be fabricated to support initially physiologic compressive loads while simultaneously supporting the formation of a neotissue.     

A multifunctional 3D co-culture system for studies of mammary tissue morphogenesis and stem cell biology

Studies on the stem cell niche and the efficacy of cancer therapeutics require complex multicellular structures and interactions between different cell types and extracellular matrix (ECM) in three dimensional (3D) space. We have engineered a 3D in vitro model of mammary gland that encompasses a defined, porous collagen/hyaluronic acid (HA) scaffold forming a physiologically relevant foundation for epithelial and adipocyte co-culture. Polarized ductal and acinar structures form within this scaffold recapitulating normal tissue morphology in the absence of reconstituted basement membrane (rBM) hydrogel. Furthermore, organoid developmental outcome can be controlled by the ratio of collagen to HA, with a higher HA concentration favouring acinar morphological development. Importantly, this culture system recapitulates the stem cell niche as primary mammary stem cells form complex organoids, emphasising the utility of this approach for developmental and tumorigenic studies using genetically altered animals or human biopsy material, and for screening cancer therapeutics for personalised medicine.     

Evaluating osteochondral defect repair potential of autologous rabbit bone marrow cells on type II collagen scaffold

The feasibility of using genipin cross-linked type II collagen scaffold with rabbit bone marrow mesenchymal stem cells (RBMSCs) to repair cartilage defect was herein studied. Induction of RBMSCs into chondrocytic phenotype on type II collagen scaffold in vitro was conducted using TGF-β 3 containing medium. After 3-weeks of induction, chondrocytic behavior, including marker genes expression and specific extracellular matrix (ECM) secretion, was observed. In the in vivo evaluation experiment, the scaffolds containing RBMSCs without prior induction were autologous implanted into the articular cartilage defects made by subchondral drilling. The repairing ability was evaluated. After 2 months, chondrocyte-like cells with lacuna structure and corresponding ECM were found in the repaired sites without apparent inflammation. After 24 weeks, we could easily find cartilage structure the same with normal cartilage in the repair site. In conclusion, it was shown that the scaffolds in combination of in vivo conditions can induce RBMSCs into chondrocytes in repaired area and would be a possible method for articular cartilage repair in clinic and cartilage tissue engineering.