Covalent attachment of a three-dimensionally printed thermoplast to a gelatin hydrogel for mechanically enhanced cartilage constructs

Hydrogels can provide a suitable environment for tissue formation by embedded cells, which makes them suitable for applications in regenerative medicine. However, hydrogels possess only limited mechanical strength, and must therefore be reinforced for applications in load-bearing conditions. In most approaches the reinforcing component and the hydrogel network have poor interactions and the synergetic effect of both materials on the mechanical properties is not effective. Therefore, in the present study, a thermoplastic polymer blend of poly(hydroxymethylglycolide-co-ε-caprolactone)/poly(ε-caprolactone) (pHMGCL/PCL) was functionalized with methacrylate groups (pMHMGCL/PCL) and covalently grafted to gelatin methacrylamide (gelMA) hydrogel through photopolymerization. The grafting resulted in an at least fivefold increase in interface-binding strength between the hydrogel and the thermoplastic polymer material. GelMA constructs were reinforced with three-dimensionally printed pHMGCL/PCL and pMHMGCL/PCL scaffolds and tested in a model for a focal articular cartilage defect. In this model, covalent bonds at the interface of the two materials resulted in constructs with an improved resistance to repeated axial and rotational forces. Moreover, chondrocytes embedded within the constructs were able to form cartilage-specific matrix both in vitro and in vivo. Thus, by grafting the interface of different materials, stronger hybrid cartilage constructs can be engineered.     

Three-dimensional perfusion culture of human adipose tissue-derived endothelial and osteoblastic progenitors generates osteogenic constructs with intrinsic vascularization capacity

In this study, we aimed at generating osteogenic and vasculogenic constructs starting from the stromal vascular fraction (SVF) of human adipose tissue as a single cell source. SVF cells from human lipoaspirates were seeded and cultured for 5 days in porous hydroxyapatite scaffolds by alternate perfusion through the scaffold pores, eliminating standard monolayer (two-dimensional [2D]) culture. The resulting cell-scaffold constructs were either enzymatically treated to extract and characterize the cells or subcutaneously implanted in nude mice for 8 weeks to assess the capacity to form bone tissue and blood vessels. SVF cells were also expanded in 2D culture for 5 days and statically loaded in the scaffolds. The SVF yielded 5.9 +/- 3.5 x 10(5) cells per milliliter of lipoaspirate containing both mesenchymal progenitors (5.2% +/- 0.9% fibroblastic colony forming units) and endothelial-lineage cells (54% +/- 6% CD34+/CD31+ cells). After 5 days, the total cell number was 1.8-fold higher in 2D than in three-dimensional (3D) cultures, but the percentage of mesenchymal- and endothelial-lineage cells was similar (i.e., 65%-72% of CD90+ cells and 7%-9% of CD34+/CD31+ cells). After implantation, constructs from both conditions contained blood vessels stained for human CD31 and CD34, functionally connected to the host vasculature. Importantly, constructs generated under 3D perfusion, and not those based on 2D-expanded cells, reproducibly formed bone tissue. In conclusion, direct perfusion of human adipose-derived cells through ceramic scaffolds establishes a 3D culture system for osteoprogenitor and endothelial cells and generates osteogenic-vasculogenic constructs. It remains to be tested whether the presence of endothelial cells accelerates construct vascularization and could thereby enhance implanted cell survival in larger size implants. Disclosure of potential conflicts of interest is found at the end of this article.     

MR assessment of osteogenic differentiation in tissue-engineered constructs

Bone marrow stromal cells (MSC) are a promising source of osteoprogenitor cells for bone tissue engineering. However, the population of the osteoprogenitor cells and their differentiation potentials change with the gender, age, and health of the donor. Development of a noninvasive method to assess osteogenic progression is critical for successful bone tissue regeneration. High-resolution magnetic resonance imaging (MRI) (at 11.7 T, with spatial resolution of 62.5 x 62.5 microm in 500 microm slices) is used in the present study to monitor osteogenic differentiation of tissue-engineered constructs prepared by seeding human bone MSCs on gelatin sponge scaffolds. Quantitative measurements of the MR relaxation times (T1, T2) and the apparent diffusion coefficient (ADC) were performed for four successive weeks on control tissue constructs and constructs exposed to osteogenic differentiation medium. The T1 and T2 relaxation times and ADC were found to decrease as osteogenic progression proceeded in samples exposed to osteogenic differentiation medium. At week 4, the T1, T2, and ADC of TE constructs were 1.81 +/- 0.11 s, 19.5 +/- 11.02 ms, and 1.01 +/- 0.47 x 10(3) mm(2)/s, respectively, for osteogenic differentiated constructs, significantly different from control constructs 2.22 +/- 0.08 s, 50.39 +/- 5.57 ms, and 1.86 +/- 0.18 x 107(3) mm(2)/s (p < 0.05). The MR parameters were also highly correlated with the cell seeding densities and alkaline phosphatase (ALP) activities of the osteogenic constructs. In conclusion, periodic measurements of MR parameters (T1, T2, and ADC) provide a promising method for noninvasive monitoring of the status of tissue-engineered bone growth and differentiation.     

Delivery of dexamethasone, ascorbate, and growth factor (TGF beta-3) in thermo-reversible hydrogel constructs embedded with rabbit chondrocytes

The aim of this study was to assess the efficacy of poly(N-isopropylacrylamide-co-acrylic acid) (p(NiPAAm-co-AAc)) as an injectable drug delivery vehicle and a cell therapeutic agent in the form of a supporting matrix for the chondrogenic differentiation of rabbit chondrocytes. The p(NiPAAm-co-AAc) hydrogel itself without specific differentiation-inducing drugs was used as a control in order to determine the effects of these materials on chondrogenic differentiation. The level of cartilage associated extracellular matrix (ECM) proteins was examined by immunohistochemical staining for collagen type II as well as Safranin-O and Alcian blue (GAG) staining. These results highlight the potential of a thermo-reversible hydrogel mixed with chondrocytes and differentiation materials as an injectable delivery vehicle for use in neocartilage formation.     

Combination of ascorbate and growth factor (TGF beta-3) in thermo-reversible hydrogel constructs embedded with rabbit chondrocytes for neocartilage formation

This study evaluated the potential of using poly(NiPAAm-co-AAc) as an injectable drug delivery carrier and a cell therapeutic agent in the form of a supporting matrix for the chondrogenic differentiation of rabbit chondrocytes. In particular, rabbit chondrocytes were embedded in hydrogels containing a combination of ascorbate and transforming growth factor beta-3 (TGF beta-3). Hydrogel constructs containing embedded cells either without ascorbate or a combination of ascorbate and TGF beta-3 were used as controls to determine the effects of ascorbate and TGF beta-3 on chondrogenic differentiation. The level of cartilage associated ECM proteins was examined using immunohistochemical staining for collagen type II as well as by Safranin-O and Alcian blue (GAG) staining. The results showed that ascorbate is an important factor for preparing cartilage constructs because of its action on chondrocyte phenotype modulation and proliferation.     

Effect of hydrogel porosity on marrow stromal cell phenotypic expression

This study describes investigation of porous photocrosslinked oligo[(polyethylene glycol) fumarate] (OPF) hydrogels as potential matrix for osteoblastic differentiation of marrow stromal cells (MSCs). The porosity and interconnectivity of porous hydrogels were assessed using magnetic resonance microscopy (MRM) as a noninvasive investigative tool that could image the water construct inside the hydrogels at a high-spatial resolution. MSCs were cultured onto the porous hydrogels and cell number was assessed using PicoGreen DNA assay. Our results showed 10% of cells initially attached to the surface of scaffolds. However, cells did not show significant proliferation over a time period of 14 days. MSCs cultured on porous hydrogels had increased alkaline phosphatase activity as well as deposition of calcium, suggesting successful differentiation and maturation to the osteoblastic phenotype. Moreover, continued expression of type I collagen and osteonectin over 14 days confirmed osteoblastic differentiation of MSCs. MRM was also applied to monitor osteogenesis of MSCs on porous hydrogels. MRM images showed porous scaffolds became consolidated with osteogenic progression of cell differentiation. These findings indicate that porous OPF scaffolds enhanced MSC differentiation leading to development of bone-like mineralized tissue.     

The synergetic effect of hydrogel stiffness and growth factor on osteogenic differentiation

Cells respond to various chemical signals as well as environmental aspects of the extracellular matrix (ECM) that may alter cellular structures and functions. Hence, better understanding of the mechanical stimuli of the matrix is essential for creating an adjuvant material that mimics the physiological environment to support cell growth and differentiation, and control the release of the growth factor. In this study, we utilized the property of transglutaminase cross-linked gelatin (TG-Gel), where modification of the mechanical properties of TG-Gel can be easily achieved by tuning the concentration of gelatin. Modifying one or more of the material parameters will result in changes of the cellular responses, including different phenotype-specific gene expressions and functional differentiations. In this study, stiffer TG-Gels itself facilitated focal contact formation and osteogenic differentiation while soft TG-Gel promoted cell proliferation. We also evaluated the interactions between a stimulating factor (i.e. BMP-2) and matrix rigidity on osteogenesis both in vitro and in vivo. The results presented in this study suggest that the interactions of chemical and physical factors in ECM scaffolds may work synergistically to enhance bone regeneration.     

Three-dimensional culture and differentiation of human osteogenic cells in an injectable hydroxypropylmethylcellulose hydrogel

The present work evaluates a newly developed silated hydroxypropylmethylcellulose (Si-HPMC)-based hydrogel as a scaffold for 3D culture of osteogenic cells. The pH variation at room temperature catalyzes the reticulation and self-hardening of the viscous polymer solution into a gelatine state. We designed reticulation time, final consistency and pH in order to obtain an easy handling matrice, suitable for in vitro culture and in vivo injection. Three human osteogenic cell lines and normal human osteogenic (HOST) cells were cultured in 3D inside this Si-HPMC hydrogel. We show here that osteosarcoma cells proliferate as clonogenic spheroids and that HOST colonies survive for at least 3 weeks. Mineralization assay and gene expression analysis of osteoblastic markers and cytokines, indicate that all the cells cultured in 3D into this hydrogel, exhibited a more mature differentiation status than cells cultured in monolayer on plastic. This study demonstrates that this Si-HPMC hydrogel is well suited to support osteoblastic survival, proliferation and differentiation when used as a new scaffold for 3D culture and represents also a potential basis for an innovative bone repair material.     

The effect of mesenchymal stromal cell-hyaluronic acid hydrogel constructs on immunophenotype of macrophages

During the past several years, multipotent mesenchymal stromal cells (MSCs) have rapidly moved from in vitro and animal studies into clinical trials as a therapeutic modality potentially applicable to a wide range of disorders. It has been proposed that ex vivo culture-expanded MSCs exert their tissue regeneration potential through their immunomodulatory and anti-inflammatory properties, and paracrine effects more than their ability to differentiate into multiple tissue lineages. Since extracellular matrix (ECM) deposition and tissue support is also one of many physiological roles of MSCs, there is increasing interest in their potential use for tissue engineering, particularly in combination with ECM-based scaffolds such as hyaluronic acid (HA). We investigated the effect of MSCs on immunophenotype of macrophages in the presence of an HA-hydrogel scaffold using a unique 3D coculture system. MSCs were encapsulated in the hydrogel and peripheral blood CD14+ monocyte-derived macrophages plated in direct contact with the MSC-gel construct. To determine the immunophenotype of macrophages, we looked at the expression of cell surface markers CD14, CD16, CD206, and human leukocyte antigen (HLA)-DR by flow cytometry. MSCs and macrophages cultured on the HA-hydrogel remained viable and were able to be recovered from the construct. There was a significant difference in the immunophenotype observed between monocyte-derived macrophages cultured on the HA scaffold compared to tissue culture polystyrene. Macrophages cultured on gels with MSCs expressed lower CD16 and HLA-DR with higher expression of CD206, indicating the least inflammatory profile overall, compatible with the immunophenotype of alternatively activated macrophages. Development of macrophages, with this immunophenotype, upon interaction with the MSC-hydrogel constructs may play a potentially significant role in tissue repair when using a cellular-biomaterial therapeutic approach.     

Material properties and osteogenic differentiation of marrow stromal cells on fiber-reinforced laminated hydrogel nanocomposites

The fibrils in the bone matrix are glued together by extracellular matrix proteins to form laminated structures (osteons) to provide elasticity and a supportive substrate for osteogenesis. The objective of this work was to investigate material properties and osteogenic differentiation of bone marrow stromal (BMS) cells seeded on osteon-mimetic fiber-reinforced hydrogel/apatite composites. Layers of electrospun poly(l-lactide) fiber mesh coated with a poly(lactide-co-ethylene oxide fumarate) (PLEOF) hydrogel precursor solution were stacked and pressed together, and crosslinked to produce a laminated fiber-reinforced composite. Hydroxyapatite (HA) nanocrystals were added to the precursor solution to produce an osteoconductive matrix for BMS cells. Acrylamide-terminated Arg–Gly–Asp (RGD) peptide (Ac-GRGD) was conjugated to the PLEOF/HA hydrogel phase to promote focal point adhesion of BMS cells. Laminates were characterized with respect to the Young’s modulus, degradation kinetics and osteogenic differentiation of BMS cells. The moduli of the laminates under dry and wet conditions were significantly higher than those of the fiber mesh and PLEOF/HA hydrogel, and within the range of values reported for wet human cancellous bone. At days 14 and 21, alkaline phosphatase (ALPase) activity of the laminates was significantly higher than those of the fiber mesh and hydrogel. Lamination significantly increased the extent of mineralization of BMS cells and laminates with HA and conjugated with RGD (Lam-RGD-HA) had 2.7-, 3.5- and 2.8-fold higher calcium content (compared to laminates without HA or RGD) after 7, 14 and 21 days, respectively. The Lam-RGD-HA group had significantly higher expression of osteopontin and osteocalcin compared to the hydrogel or laminates without HA or RGD, consistent with the higher ALPase activity and calcium content of Lam-RGD-HA. Laminated osteon-mimetic structures have the potential to provide mechanical strength to the regenerating region as well as supporting the differentiation of progenitor cells to the osteogenic lineage.     

Structural and molecular micropatterning of dual hydrogel constructs for neural growth models using photochemical strategies

Chemotactic and haptotactic cues guide neurite growth toward appropriate targets by eliciting attractive or repulsive responses from the neurite growth cones. Here we present an integrated system allowing both structural and molecular micropatterning in dual hydrogel 3D tissue culture constructs for directing in vitro neuronal growth via structural, immobilized, and soluble guidance cues. These tissue culture constructs were fabricated into specifiable geometries using UV light reflected from a digital micromirror device acting as a dynamic photomask, resulting in dual hydrogel constructs consisting of a cell growth-restrictive polyethylene glycol (PEG) boundary with a cell growth-permissive interior of photolabile α-carboxy-2-nitrobenzyl cysteine agarose (CNBC-A). This CNBC-A was irradiated in discrete areas and subsequently tagged with maleimide-conjugated biomolecules. Fluorescent microscopy showed biomolecule binding only at the sites of irradiation in CNBC-A, and confocal microscopy confirmed 3D binding through the depth of the construct. Neurite outgrowth studies showed contained growth throughout CNBC-A. The diffusion rate of soluble fluorescein-bovine serum albumin through the dual hydrogel construct was controlled by PEG concentration and the distance between the protein source and the agarose interior; the timescale for a transient protein gradient changed with these parameters. These findings suggest the dual hydrogel system is a useful platform for manipulating a 3D in vitro microenvironment with patterned structural and molecular guidance cues for modeling neural growth and guidance.     

Bioimaging of dexamethasone and TGF beta-1 and its biological activities of chondrogenic differentiation in hydrogel constructs

This study examined the efficacy of poly(NiPAAm-co-AAc) as an injectable drug delivery vehicle and a cell therapeutic agent in the form of a supporting matrix for the chondrogenic differentiation of rabbit chondrocytes. The hydrogel constructs, which consisted of embedded cells co-encapsulating dexamethasone (Dex) and TGF beta-1 or unloaded Dex, were used as controls to determine the effects of Dex and TGF beta-1 on chondrogenic differentiation. The level of Dex and TGF beta-1 released was monitored using a bioimaging method. The amount of Dex released from hydrogel was faster than that of TGF beta-1. TGF beta-1 was present in hydrogel for more than 4 weeks after the injection. The level of the cartilage associated ECM proteins was examined by immunohistochemical staining for collagen type II as well as by Safranin-O and Alcian blue (GAG) staining. These results highlight the potential of a thermo-reversible hydrogel mixed with the chondrocytes and differentiation delivery material for applications in neocartilage formation.     

Combination material delivery of dexamethasone and growth factor in hydrogel blended with hyaluronic acid constructs for neocartilage formation

The aim of this study was to assess the efficacy of poly(NiPAAm-co-AAc) blended with hyaluronic acid (HA) as an injectable cell vehicle and a cell therapeutic agent in the form of a supporting matrix for the chondrogenic differentiation of rabbit chondrocytes. Specially, rabbit chondrocytes were embedded in blended hydrogels co-encapsulation with dexamethasone (Dex) and growth factors for enhancing the chondrogenic differentiation. Blended hydrogel constructs consisting of embedded cells co-encapsulating Dex and TGF beta-3 or unloaded Dex and sTGF beta-3 served as controls to assess the effects of Dex on chondrogenic differentiation. Hydrogel constructs consisting of embedded cells co-encapsulating Dex and TGF beta-3 on chondrogenic differentiation. The hydrogel constructs were injected subcutaneously into the nude mice and monitored for 1, 4, and 8 weeks after the injection. The level of the cartilage-associated ECM proteins was determined by immunohistochemical (collagen type II; specific marker for chondrogenic differentiation), Safranin-O, and Alcian blue (GAG) staining. Over the same time period, the glycosamingoglycan content per cell remained constant for all formulations, indicating that the dramatic increase in cell number for samples with Dex and TGF beta-3 loaded in hydrogel constructs was accompanied by maintenance of the cell phenotypes.     

Engineering of large osteogenic grafts with rapid engraftment capacity using mesenchymal and endothelial progenitors from human adipose tissue

We investigated whether the maintenance in culture of endothelial and mesenchymal progenitors from the stromal vascular fraction (SVF) of human adipose tissue supports the formation of vascular structures in vitro and thereby improves the efficiency and uniformity of bone tissue formation in vivo within critically sized scaffolds. Freshly-isolated human SVF cells were seeded and cultured into hydroxyapatite scaffolds (1 cm-diameter, 1 cm-thickness) using a perfusion-based bioreactor system, which resulted in maintenance of CD34(+)/CD31(+) endothelial lineage cells. Monolayer-expanded isogenic adipose stromal cells (ASC) and age-matched bone marrow stromal cells (BMSC), both lacking vasculogenic cells, were used as controls. After 5 days in vitro, SVF-derived endothelial and mesenchymal progenitors formed capillary networks, which anastomosed with the host vasculature already 1 week after ectopic nude rat implantation. As compared to BMSC and ASC, SVF-derived cells promoted faster tissue ingrowth, more abundant and uniform bone tissue formation, with ossicles reaching a 3.5 mm depth from the scaffold periphery after 8 weeks. Our findings demonstrate that maintenance of endothelial/mesenchymal SVF cell fractions is crucial to generate osteogenic constructs with enhanced engraftment capacity. The single, easily accessible cell source and streamlined, bioreactor-based process makes the approach attractive towards manufacturing of clinically relevant sized bone substitute grafts.     

不同材料因素影响骨髓间充质干细胞的增殖及成骨分化

文题释义：表面微观形貌：材料表面所具有的所有微观几何形状及相关物理量统称为表面微观形貌,包括孔径大小、孔径率、表面凹凸程度、底物刚度、弹性模量、表面能等方面,同一种材料不同的表面微观形貌将会对材料表面的物质产生不同的影响。 表面能/润湿性：表面能是指恒温、恒压、恒组成情况下可逆地增加物系表面积须对物质所做的非体积功,润湿性是指一种液体在一种固体表面铺展的能力或倾向性。 背景：在利用组织工程技术治疗骨缺损或骨损伤的过程中,生物材料对移植的骨髓间充质干细存活率与增殖分化潜能有影响。 目的：对生物材料如何影响骨髓间充质干细胞的增殖及成骨分化的研究进展进行综述,指导材料合理应用。 方法：应用计算机检索中国知网、PubMed、Web of science及万方数据库,检索词为“骨髓间充质干细胞、成骨分化、生物材料、微观形貌、Bone marrow mesenchymal stem cells、Osteogenic differentiation、Biological materials、The microstructure”,根据标准纳入不同生物材料因素影响骨髓间充质干细胞增殖及成骨分化的文献。结果与结论：①金属材料具有良好的生物相容性、骨传导性、机械性能,非金属材料具有良好的生物相容性、骨传导性、再吸收性及三维塑形性;②材料的表面微观形貌因素众多,即使是同一种材料,表面能/润湿性越高越利于骨髓间充质干细胞的增殖与成骨分化,表面粗糙程度相对越高越利于细胞的增殖、黏附及分化,孔径直径越大、孔径率越小越利于细胞的成骨分化,底物刚度相对高及弹性模量相对大有利于骨髓间充质干细胞的成骨分化,以上材料的各种因素等都能够影响骨髓间充质干细胞的增殖及成骨作用,这些新的研究进展进一步提高了骨髓间充质干细胞种植于各类生物材料用于临床治疗的应用。ORCID: 0000-0001-8325-9867(王旸昊) 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

The integration of 3-D cell printing and mesoscopic fluorescence molecular tomography of vascular constructs within thick hydrogel scaffolds

Developing methods that provide adequate vascular perfusion is an important step toward engineering large functional tissues. Meanwhile, an imaging modality to assess the three-dimensional (3-D) structures and functions of the vascular channels is lacking for thick matrices (>2 ≈ 3 mm). Herein, we report on an original approach to construct and image 3-D dynamically perfused vascular structures in thick hydrogel scaffolds. In this work, we integrated a robotic 3-D cell printing technology with a mesoscopic fluorescence molecular tomography imaging system, and demonstrated the capability of the platform to construct perfused collagen scaffolds with endothelial lining and to image both the fluid flow and fluorescent-labeled living endothelial cells at high-frame rates, with high sensitivity and accuracy. These results establish the potential of integrating both 3-D cell printing and fluorescence mesoscopic imaging for functional and molecular studies in complex tissue-engineered tissues.     

Effect of the fiber diameter and porosity of non-woven PET fabrics on the osteogenic differentiation of mesenchymal stem cells

The proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) was investigated in three-dimensional non-woven fabrics prepared from polyethylene terephthalate (PET) fiber with different diameters. When seeded into the fabrics of cell scaffold, more MSC attached in the fabric of thicker PET fibers than that of thinner ones, irrespective of the fabric porosity. The morphology of cells attached became more spreaded with an increase in the fiber diameter of fabrics. The rate of MSC proliferation depended on the PET fiber diameter and porosity of fabrics: the bigger the fiber diameter of fabrics with higher porosity, the higher their proliferation rate. When the alkaline phosphatase (ALP) activity and osteocalcin content of MSC cultured in different types of fabrics was measured to evaluate the ostegenic differentiation, they became maximum for the non-woven fabrics with a fiber diameter of 9.0 microm, although the values of low-porous fabrics were significantly high compared with those of high porous fabrics. We concluded that the attachment, proliferation and bone differentiation of MSC was influenced by the fiber diameter and porosity of non-woven fabrics as the scaffold.