Likun Guo

An in vitro study of collagen hydrogel to induce the chondrogenic differentiation of mesenchymal stem cells

It is controversial whether a biomaterial itself, rather than addition of any exogenous growth factor, could induce mesenchymal stem cells (MSCs) to differentiate into chondrogenic lineage, further to regenerate cartilage. Previous studies have shown that collagen-based hydrogel could induce MSCs to differentiate into chondrocytes in vivo but the in vitro studies only have a few reports. The evidence that biomaterials could induce chondrogenesis is not adequate. In this study, we tried to address whether type I collagen hydrogel has chondro-inductive capability in vitro and how this scaffold induces MSCs to generate cartilage tissue without exogenous growth factors in the culture medium. We encapsulated neonatal rabbit bone marrow mesenchymal stem cells (BMSCs) in type I collagen hydrogel homogeneously or implanted cell aggregates in hydrogel, and cultured them in nonchondrogenic inductive media. After at least 28 days culture, cells in the homogeneous group were tending to chondrogenic differentiation while cell density was high, and cells in the aggregate group have almost gone through chondrogenesis and formed neo-cartilage tissue with abundant specific extracellular matrix (ECM) deposition. These results indicate collagen hydrogel has inherent inductivity for the chondrogenic differentiation of BMSCs, and the optimum specification and tissue formation were accompanied with local high cell density. This research suggests a feasible strategy to induce the chondro differentiation of BMSCs independent of exogenous growth factors, which may greatly contribute to clinical cartilage regeneration.

Osteogenic differentiation of human mesenchymal stem cells on chargeable polymer-modified surfaces.

Polystyrene cell-culture plates modified with positively charged polyallylamine (PAAm) and negatively charged poly(acrylic acid) (PAAc) and unmodified plate were used for the culture of human mesenchymal stem cells (MSCs) to study the effect of surface electrostatic properties on their osteogenic differentiation. All of these surfaces supported cell adhesion and proliferation. However, the cells adhered, spread, and proliferated somewhat more quickly on the PAAm-modified surface than they did on the PAAc-modified and control surfaces. Osteogenic differentiation was examined by alkaline phosphatase (ALP) staining, alizarin red S staining, and gene-expression analysis. The MSCs cultured on the three kinds of surfaces in the presence of dexamethasone were positively stained with ALP and alizarin red S staining, while the cells cultured without dexamethasone were not positively stained. Gene-expression analyses using real-time PCR indicated that MSCs cultured on these surfaces in the presence of dexamethasone expressed osteogenic marker genes, encoding ALP, osteocalcin, bone sialoprotein, osteopontin, and type I collagen. These results indicate that the positively charged, negatively charged, and unmodified surfaces supported osteogenic differentiation, and that their effect required the synergistic effect of dexamethasone.

Photo-cross-linkable methacrylated gelatin and hydroxyapatite hybrid hydrogel for modularly engineering biomimetic osteon.

Modular tissue engineering holds great potential in regenerating natural complex tissues by engineering three-dimensional modular scaffolds with predefined geometry and biological characters. In modular tissue-like construction, a scaffold with an appropriate mechanical rigidity for assembling fabrication and high biocompatibility for cell survival is the key to the successful bioconstruction. In this work, a series of composite hydrogels (GH0, GH1, GH2, and GH3) based on a combination of methacrylated gelatin (GelMA) and hydroxyapatite (HA) was exploited to enhance hydrogel mechanical rigidity and promote cell functional expression for osteon biofabrication. These composite hydrogels presented a lower swelling ratio, higher mechanical moduli, and better biocompatibility when compared to the pure GelMA hydrogel. Furthermore, on the basis of the composite hydrogel and photolithograph technology, we successfully constructed an osteon-like concentric double-ring structure in which the inner ring encapsulating human umbilical vascular endothelial cells (HUVECs) was designed to imitate blood vessel tubule while the outer ring encapsulating human osteoblast-like cells (MG63s) acts as part of bone. During the coculture period, MG63s and HUVECs exhibited not only satisfying growth status but also the enhanced genic expression of osteogenesis-related and angiogenesis-related differentiations. These results demonstrate this GelMA-HA composite hydrogel system is promising for modular tissue engineering.

Modulation of immunological properties of allogeneic mesenchymal stem cells by collagen scaffolds in cartilage tissue engineering.

Influence of the structures of some collagen scaffolds on immunological properties of the seeded allogeneic mesenchymal stem cells (MSCs) was studied in this article. Hydrogels, sponge, and membrane were prepared from type-I collagen. These scaffolds were seeded with neonatal rabbit MSCs and cultured for different periods. Changes of the immunological properties associated with different scaffolds were analyzed and compared. It was found that the expression of major histocompatibility complex (MHC) class I and II molecules on MSCs increased gradually in all scaffolds, but the least increment was recorded in hydrogels. Mixed lymphocyte reactions (MLR) showed that the MSC-hydrogel constructs invoked considerably low allogeneic lymphocytes proliferation. Even in presence of interferon-γ (IFN-γ), the hydrogels with higher concentration gave comparatively lower increment of MHC-II expression and allogeneic lymphocytes proliferation. These results suggest that different scaffold structures may provide different microenvironments and extents of isolation from the host immune system for the seed cells, thereby affecting their immunological properties. Therefore, scaffold structures may modulate the immunological properties of tissue-engineered cartilage with allogeneic cells. Hydrogels, especially which were prepared from higher collagen concentrations, were found to be a promising scaffold structure, from the perspective of avoiding severe immune rejection.

Effects of Composition and Mechanical Property of Injectable Collagen I/II Composite Hydrogels on Chondrocyte Behaviors.

Satisfactory repair of damaged articular cartilage is still a challenge, while tissue engineering provides a promising strategy. Collagen-based hydrogels have been widely applied in cartilage tissue engineering due to their biocompatibility. In this study, type I collagen and type II collagen were selected to prepare physically crosslinked composite hydrogels by self-assembly of collagen, and the effects of their physicochemical properties on chondrocyte phenotype maintenance and extracellular matrix (ECM) secretion were investigated. First, the microstructure of hydrogels was observed by a scanning electron microscope, and the compressive modulus was measured by a dynamic mechanical analyzer. Then, chondrocytes were encapsulated in hydrogels and detected by Live/Dead staining. The secretion of ECM was qualitatively estimated by histological staining and quantitatively analyzed by sulfated glycosaminoglycans and DNA content detection. Finally, cartilage-specific gene expression was analyzed by quantitative real-time polymerase chain reaction analysis. The results showed that the microstructure and mechanical property of hydrogels were relevant to the composition of composite hydrogels. The compressive modulus of hydrogels improved with the increase of type I collagen content in the hydrogels. Chondrocytes could maintain their round or oval morphology and secrete cartilage-specific ECM in the four groups of hydrogels, but higher the compressive modulus of composite hydrogels, the more ECM secretion of chondrocytes.

Preparation and characterization of macromolecule cross-linked collagen hydrogels for chondrocyte delivery.

Collagen hydrogels are widely used in cartilage tissue engineering for their mimicked chondrogenic environment. Due to the rapid degradation nature and weak mechanical property, collagen hydrogels are often cross-linked in application. In this work, collagen hydrogels were soaked into oxidized alginate solution which used as macromolecular cross-linker to prepare the cross-linked hydrogels. Soaking method could retain the self-assemble property of collagen and also bring in a cross-linking network. The compressive modulus and degradation properties of collagen hydrogels were ameliorated after cross-linked, and chondrocytes encapsulated in the cross-linked hydrogels proliferated well and maintained the cell phenotype. This study implied that collagen hydrogels cross-linked by oxidized alginate may have a great potential for application in cartilage tissue engineering.

Photo-crosslinked mono-component type II collagen hydrogel as a matrix to induce chondrogenic differentiation of bone marrow mesenchymal stem cells

Type II collagen is a prospective chondro-inductive matrix for bone marrow mesenchymal stem cells (BMSCs), a key component of the extracellular matrix of cartilage; however, its application is limited by deficient fibrillogenesis and gelation. Herein, type II collagen methacrylamide (Col-II-MA) was synthesized by an amidation reaction between the ε-amino groups on collagen lysine and methacrylic anhydride to enable photo-crosslinking of the collagen, thus accomplishing a one-step preparation of mono-component type II collagen hydrogel for the first time. BMSCs encapsulated within the Col-II-MA hydrogel exhibited accelerated proliferation and morphological changes that are similar to chondrogenesis, as well as up-regulated expression of chondrogenic genes and remarkable secretion of the cartilaginous matrix. These results demonstrated that this effective synthetic approach facilitated the formation of photo-active type II collagen hydrogel with a well-preserved triple helical conformation, which provides BMSCs with a favorable microenvironment for growth and the essential chondro-inductive matrix for differentiation. Furthermore, the hydrogel is applicable to microfabrication techniques and displays promise for future applications in microscale tissue engineering.

Fabrication of gelatin-micropatterned surface and its effect on osteogenic differentiation of hMSCs

Studies of the effect of surface chemistries on cell functions on a single surface with different surface chemistry areas have been achieved by micropatterning technology. In this study, a gelatin-micropatterned surface was prepared and used for direct comparison of the effect of different surface chemistries on the osteogenic differentiation of stem cells under the same culture environment. The micropattern was prepared by UV-irradiating photo-reactive azidobenzoyl-derived gelatin-coated surface on a polystyrene plate through a photomask with 200 μm-width stripes. Human bone marrow-derived mesenchymal stem cells (hMSCs) were cultured on the gelatin-modified or micropatterned surfaces in osteogenic differentiation induction medium. Alkaline phosphatase (ALP) staining, alizarin red S staining and real-time PCR analysis were performed to detect the effect of surface chemistry on the osteogenic differentiation of hMSCs. The cells on both the polystyrene area and the gelatin-micropatterned area showed positive ALP staining. Alizarin red S staining results showed that the gelatin-micropatterned area promoted the deposition of minerals. Real-time PCR results indicated that the gelatin-modified surface promoted the expression of osteogenic specific genes encoding ALP and bone sialoprotein. The results suggested the promotive effect of gelatin on the osteogenic differentiation of hMSCs.

Fabrication and characterization of collagen-based injectable and self-crosslinkable hydrogels for cell encapsulation

Injectable and self-crosslinkable hydrogels have drawn much attention for their potential application as cell delivery carriers to deliver cells to the injury site of arbitrary shape. In this study, injectable and self-crosslinkable hydrogels were designed and fabricated based on collagen type I (Col I) and activated chondroitin sulfate (CS-sNHS) by physical and chemical crosslinking without the addition of any catalysts. The physical properties of hydrogels, including mechanical properties, swelling and degradation properties, were investigated. The results demonstrated that the physical properties of hydrogels, especially the stiffness of hydrogels, were readily tuned by varying the degree of substitution (DS) of CS-sNHS without changing the concentration of collagen-based precursor. Chondrocytes were encapsulated into hydrogels to investigate the effects of hydrogels on the survival, proliferation and extracellular matrix (ECM) secretion of cells by FDA/PI staining, CCK-8 test and histological staining. The results suggested that all of these hydrogels supported the survival and ECM secretion of chondrocytes, while there was more ECM secretion around chondrocytes encapsulated in hydrogel Col I/CS-sNHS56% in which the DS of CS-sNHS was 56%. When the neutral precursor solution for hydrogel of Col I or Col I/CS-sNHS56% was subcutaneously injected into SD rats, hydrogels both displayed acceptable biocompatibility in vivo. These results imply that these injectable and self-crosslinkable hydrogels are suitable candidates for applications in the fields of cell delivery and tissue engineering.

A Novel Way to Prepare Nano-Hydroxyapatite/Poly(D,L-Lactide) Composite

A composite of needle-like nano-hydroxyapatite (n-HA)/poly (D,L) lactide (PDLLA) was prepared. The n-HA crystals were poorly crystallized and uniformly distributed in the composite with a crystal size of 10–20 nm in diameter by 40–60nm in length，which was smaller than that of pure nano-HA. Molecular interactions and chemical bonds might present between n-HA and PDLLA in the composite, which were revealed by IR and XPS. The synthetic n-HA/PDLLA composite had a good homogeneity and could be a bioactive material for bone defect especially for load-bearing bone repair, which is more potential than pure HA or pure PDLLA.