A photochemical crosslinking technology for tissue engineering - Enhancement of the physico-chemical properties of collagen-based scaffolds

Collagen gel is a natural biomaterial commonly used in tissue engineering because of its close resemblance to nature, negligible immunogenecity and excellent biocompatibility. However, unprocessed collagen gel is mechanically weak, highly water binding and vulnerable to chemical and enzymatic attacks that limits its use in tissue engineering in particular tissues for weight-bearing purposes. The current project aimed to strengthen and stabilize collagen scaffolds using a photochemical crosslinking technique. Photochemical crosslinking is rapid, efficient, non-thermal and does not involve toxic chemicals, comparing with other crosslinking methods such as glutaraldehyde and gamma irradiation. Collagen scaffolds were fabricated using rat-tail tendon collagen. An argon laser was used to process the collagen gel after equilibrating with a photosensitizing reagent. Scanning electronic microscope was used to characterize the surface and cross-sectional morphology of the membranes. Physico-chemical properties of the collagen scaffolds such as water-binding capacity, mechanical properties and thermostability were studied. Photochemical crosslinking significantly reduced the water-binding capacity, a parameter inversely proportional to the extent of crosslinking, of collagen scaffolds. Photochemical crosslinking also significantly increased the ultimate stress and tangent modulus at 90% of the rupture strain of the collagen scaffolds. Differential scanning calorimetry analysis showed a significantly higher shrinkage temperature and absence of the denaturation peak during the thermoscan comparing with the controls. This means greater thermostability in the photochemically crosslinked collagen scaffolds. This study demonstrates that the photochemical crosslinking technology is able to enhance the physicochemical properties of collagen scaffolds by strengthening, stabilizing and controlling the swelling ratio of the collagen scaffolds so as to enable their use for tissue engineering.published_or_final_versio

Biomedical applications of photochemistry

Photochemistry is the study of photochemical reactions between light and molecules. Recently, there have been increasing interests in using photochemical reactions in the fields of biomaterials and tissue engineering. This work revisits the components and mechanisms of photochemistry and reviews biomedical applications of photochemistry in various disciplines, including oncology, molecular biology, and biosurgery, with particular emphasis on tissue engineering. Finally, potential toxicities and research opportunities in this field are discussed. © 2010 Mary Ann Liebert, Inc.published_or_final_versio

Collagen scaffold remodeling by human mesenchymal stem cells

DMM 2011 entitled: Re-engineering Regenerative MedicinePoster Session - Tissue Engineering: no. 82Type I collagen has been widely used as scaffold for tissue engineering because of its excellent biocompatibility and negligible immunogenicity. We previously have developed a collagen microencapsulation technology entrapping many cells including human mesenchymal stem cells (hMSCs) in microspheres made of nanofibrous collagen meshwork. Nevertheless, little is understood about how stem cells interact with and remodel the collagen meshwork. This study aims to investigate collagen remodeling by human mesenchymal stem cells (hMSCs) in terms of degradation, new matrix synthesis, and re‐organization. We …postprin

A structurally and functionally biomimetic biphasic scaffold for intervertebral disc tissue engineering.

Tissue engineering offers high hopes for the treatment of intervertebral disc (IVD) degeneration. Whereas scaffolds of the disc nucleus and annulus have been extensively studied, a truly biomimetic and mechanically functional biphasic scaffold using naturally occurring extracellular matrix is yet to be developed. Here, a biphasic scaffold was fabricated with collagen and glycosaminoglycans (GAGs), two of the most abundant extracellular matrix components in the IVD. Following fabrication, the scaffold was characterized and benchmarked against native disc. The biphasic scaffold was composed of a collagen-GAG co-precipitate making up the nucleus pulposus-like core, and this was encapsulated in multiple lamellae of photochemically crosslinked collagen membranes comprising the annulus fibrosus-like lamellae. On mechanical testing, the height of our engineered disc recovered by similar to 82-89% in an annulus-independent manner, when compared with the 99% recovery exhibited by native disc. The annulus-independent nature of disc height recovery suggests that the fluid replacement function of the engineered nucleus pulposus core might mimic this hitherto unique feature of native disc. Biphasic scaffolds comprised of 10 annulus fibrosus-like lamellae had the best overall mechanical performance among the various designs owing to their similarity to native disc in most aspects, including elastic compliance during creep and recovery, and viscous compliance during recovery. However, the dynamic mechanical performance (including dynamic stiffness and damping factor) of all the biphasic scaffolds was similar to that of the native discs. This study contributes to the rationalized design and development of a biomimetic and mechanically viable biphasic scaffold for IVD tissue engineering.published_or_final_versio

Effect of dynamic mechanical compression on actin cytoskeleton network of human mesenchymal stem cells (hMSCs) in three dimensional collagen constructs

Actin filament, one type of cytoskeletons, plays a central role in mediating cellular in responses to mechanical loading. Many mechanorgulation studies are restricted to 2D models using isolated cells or monolayer cultures, even though it is know that cells behave differently in term of cell morphology, cell matrix adhesion composition and matrix mediated force transmission when they are in 3D configuration. This current study investigates the temporal change of actin network of hMSCs entrapped in 3D collagen construct upon cyclic compression. Human bone marrow mesenchymal stem cells were encapsulated in cylindrical collagen construct. A micromanipulator based loading device coupled to fluorescent microscope was used to deliver compression loading to the construct with 10% strain at 1Hz for different period of time. Rhodamine phalloidin was used to stain for the actin filament network to hMSC in the construct at different time points postcompression. An optimized loading protocol with 5hrs of continuous loading was delivered. Actin network concentrated at the cell periphery of cells exhibiting round morphology was observed immediately while elongated and polarized actin network was found after 24 hours. Detailed characterization of actin filament organization and their association with cell-matrix interaction molecules are warrented before the mechanisms of compression-induced hMSC alignment can be delineated © 2010 IEEE.published_or_final_versionThe IEEE 4th International Conference on Nano/Molecular Medicine and Engineering (NANOMED 2010), Hong Kong/Macau, China, 5-9 December 2010. In Proceedings of the IEEE International Conference on NANOMED, 2010, p. 136-13

3D matrix adhesions mediating mechanostranduction in hMSC-collagen constructs

The current study aims to identify the type of cell-matrix adhesions of hMSCs in 3D collagen constructs and to investigate the effects of dynamic compression on the type, morphology and composition of cell-matrix adhesions, particularly to observe whether the compression stimulates the maturation or evolvement of 3D matrix adhesion in hMSC-collagen constructs. Preliminary results demonstrated the colocalization of integrin α 5 β 1 and fibronectin in cell-matrix adhesions in loaded constructs, partially fulfilling the requirements for 3D matrix adhesion to evolve. In addition, fibronectin was shown to be organized into tiny-dotted adhesions in loaded constructs in a loading duration dependent way, suggesting dynamic compression may be able to mature adhesions in the constructs, hopefully into 3D matrix adhesions. It was also demonstrated that hMSCs plated onto their own cell-derived matrices form elongated adhesions which are similar to 3D matrix adhesions formed by fibroblasts. Further characterization on the cell-matrix adhesions of hMSCs in 3D collagen constructs and identification of differences in adhesions between loaded and unloaded constructs are underway. © 2010 IEEE.published_or_final_versionThe IEEE 4th International Conference on Nano/Molecular Medicine and Engineering (NANOMED 2010), Hong Kong/Macau, China, 5-9 December 2010. In Proceedings of the IEEE International Conference on NANOMED, 2010, p. 34-3

Effects of reconstituted collagen matrix on fates of mouse embryonic stem cells before and after induction for chondrogenic differentiation

Embryonic stem (ES) cells are pluripotent cells with great potential in regenerative medicine. However, controlling their differentiation toward homogeneous lineages is challenging. In this study, we aim to investigate the effects of reconstituted 3D collagen matrix on the fates of mouse ES (mES) cells before and after induction for chondrogenic differentiation. Specifically, mES cells were encapsulated and cultured in 3D collagen microspheres and exposed to induction signals at different time points. Growth characteristics and differentiation status of mES cells were then evaluated. Collagen microspheres provided a suitable microenvironment supporting mES cell growth and maintaining their undifferentiated status for certain period of time. At later time points, the proportion of undifferentiated mES cells gradually decreased, accompanied by increasing proportions of mesenchymal progenitor cells. This suggests the inductive role of collagen matrix in differentiating mES cells toward mesenchymal lineages. Moreover, a lower initial collagen monomer concentration facilitated the differentiation of mES cells into chondrogenic lineages, while induction at a later time point associated with a more advanced stage of chondrogenic differentiation. This indicates that both the initial collagen concentration and the time to induce differentiation significantly affected the fates of mES cells. This study contributes to future development of ES cell-based therapies. © Copyright 2009, Mary Ann Liebert, Inc.published_or_final_versionThe 7th Annual Meeting of the International Society for Stem Cell Research (ISSCR), Barcelona, Spain, 8-11 July 2009. In Tissue Engineering. Part A, 2009, v. 15 n. 10, p. 3071-308

Microencapsulation of Neuroblastoma Cells and Mesenchymal Stromal Cells in Collagen Microspheres: A 3D Model for Cancer Cell Niche Study

There is a growing trend for researchers to use in vitro 3D models in cancer studies, as they can better recapitulate the complex in vivo situation. And the fact that the progression and development of tumor are closely associated to its stromal microenvironment has been increasingly recognized. The establishment of such tumor supportive niche is vital in understanding tumor progress and metastasis. The mesenchymal origin of many cells residing in the cancer niche provides the rationale to include MSCs in mimicking the niche in neuroblastoma. Here we co-encapsulate and co-culture NBCs and MSCs in a 3D in vitro model and investigate the morphology, growth kinetics and matrix remodeling in the reconstituted stromal environment. Results showed that the incorporation of MSCs in the model lead to accelerated growth of cancer cells as well as recapitulation of at least partially the tumor microenvironment in vivo. The current study therefore demonstrates the feasibility for the collagen microsphere to act as a 3D in vitro cancer model for various topics in cancer studies.published_or_final_versio

Extracellular Protease Inhibition Alters the Phenotype of Chondrogenically Differentiating Human Mesenchymal Stem Cells (MSCs) in 3D Collagen Microspheres

Matrix remodeling of cells is highly regulated by proteases and their inhibitors. Nevertheless, how would the chondrogenesis of mesenchymal stem cells (MSCs) be affected, when the balance of the matrix remodeling is disturbed by inhibiting matrix proteases, is incompletely known. Using a previously developed collagen microencapsulation platform, we investigated whether exposing chondrogenically differentiating MSCs to intracellular and extracellular protease inhibitors will affect the extracellular matrix remodeling and hence the outcomes of chondrogenesis. Results showed that inhibition of matrix proteases particularly the extracellular ones favors the phenotype of fibrocartilage rather than hyaline cartilage in chondrogenically differentiating hMSCs by upregulating type I collagen protein deposition and type II collagen gene expression without significantly altering the hypertrophic markers at gene level. This study suggests the potential of manipulating extracellular proteases to alter the outcomes of hMSC chondrogenesis, contributing to future development of differentiation protocols for fibrocartilage tissues for intervertebral disc and meniscus tissue engineering.published_or_final_versio

Carriers in Cell-Based Therapies for Neurological Disorders

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