Stephen D. Waldman

Mesenchymal progenitor self-renewal deficiency leads to age-dependent osteoporosis in Sca-1/Ly-6A null mice

The cellular and molecular mechanisms that underlie age-dependent osteoporosis, the most common disease in the Western Hemisphere, are poorly understood in part due to the lack of appropriate animal models in which to study disease progression. Here, we present a model that shows many similarities to the human disease. Sca-1, well known for its expression on hematopoietic stem cells, is present on a subset of bone marrow stromal cells, which potentially include mesenchymal stem cells. Longitudinal studies showed that Sca-1−/− mice undergo normal bone development but with age exhibit dramatically decreased bone mass resulting in brittle bones. In vivo and in vitro analyses demonstrated that Sca-1 is required directly for the self-renewal of mesenchymal progenitors and indirectly for the regulation of osteoclast differentiation. Thus, defective mesenchymal stem or progenitor cell self-renewal may represent a previously uncharacterized mechanism of age-dependent osteoporosis in humans.

Effect of Biomechanical Conditioning on Cartilaginous Tissue Formation in Vitro

Background: Although tissue engineering of articular cartilage is a promising approach for cartilage repair, it has been difficult to develop cartilaginous tissue in vitro that mimics the properties of native cartilage. Isolated chondrocytes grown in culture typically do not accumulate enough extracellular matrix, and the generated tissue possesses only a fraction of the mechanical properties of native cartilage. One potential explanation for this might be that the cells are grown in an environment that lacks the mechanical stimuli to which the chondrocytes are exposed in vivo. In this study, we compared the long-term effects of both dynamic compressive and shearing forces on cartilaginous tissue formation in vitro.Methods: Bovine articular chondrocytes were grown on the surface of porous ceramic substrates and were maintained under static, free-swelling conditions for a period of four weeks. Cultures were then subjected to six minutes of mechanical stimulation every other day, in either compression or shear, for an additional four-week period.Results: Cartilaginous tissues cultured in the presence of intermittent compression or shear were significantly thicker (p < 0.05) and had accumulated more extracellular matrix (p < 0.01) compared with the unstimulated controls. However, when normalized by the wet weight of the tissue, cultures stimulated in the presence of shearing forces contained more proteoglycans and collagen compared with compression-stimulated cultures. These cultures also displayed the largest increase in mechanical properties, with a threefold increase in equilibrium stress and a fivefold increase in equilibrium modulus.Conclusions and Clinical Relevance: The results of this study demonstrate that a brief application of mechanical forces applied periodically over a long duration can improve the quality of cartilaginous tissue formed in vitro. However, the changes in tissue composition and mechanical properties were dependent on the specific mode of the applied mechanical forces, with shear stimulation eliciting the greater effect. This finding suggests that chondrocytes may respond differently to different modes of applied forces.

Genipin Cross-Linked Fibrin Hydrogels for in vitro Human Articular Cartilage Tissue-Engineered Regeneration

Our objective was to examine the potential of a genipin cross-linked human fibrin hydrogel system as a scaffold for articular cartilage tissue engineering. Human articular chondrocytes were incorporated into modified human fibrin gels and evaluated for mechanical properties, cell viability, gene expression, extracellular matrix production and subcutaneous biodegradation. Genipin, a naturally occurring compound used in the treatment of inflammation, was used as a cross-linker. Genipin cross-linking did not significantly affect cell viability, but significantly increased the dynamic compression and shear moduli of the hydrogel. The ratio of the change in collagen II versus collagen I expression increased more than 8-fold over 5 weeks as detected with real-time RT-PCR. Accumulation of collagen II and aggrecan in hydrogel extracellular matrix was observed after 5 weeks in cell culture. Overall, our results indicate that genipin appeared to inhibit the inflammatory reaction observed 3 weeks after subcutaneous implantation of the fibrin into rats. Therefore, genipin cross-linked fibrin hydrogels can be used as cell-compatible tissue engineering scaffolds for articular cartilage regeneration, for utility in autologous treatments that eliminate the risk of tissue rejection and viral infection.

Design and characterization of a biodegradable composite scaffold for ligament tissue engineering

Herein we report on the development and characterization of a biodegradable composite scaffold for ligament tissue engineering based on the fundamental morphological features of the native ligament. An aligned fibrous component was used to mimic the fibrous collagen network and a hydrogel component to mimic the proteoglycan-water matrix of the ligament. The composite scaffold was constructed from cell-adherent, base-etched, electrospun poly(epsilon-caprolactone-co-D,L-lactide) (PCLDLLA) fibers embedded in a noncell-adherent photocrosslinked N-methacrylated glycol chitosan (MGC) hydrogel seeded with primary ligament fibroblasts. Base etching improved cellular adhesion to the PCLDLLA material. Cells within the MGC hydrogel remained viable (72 +/- 4%) during the 4-week culture period. Immunohistochemistry staining revealed ligament ECM markers collagen type I, collagen type III, and decorin organizing and accumulating along the PCLDLLA fibers within the composite scaffolds. On the basis of these results, it was determined that the composite scaffold design was a viable alternative to the current approaches used for ligament tissue engineering and merits further study.

Biomimetic poly(lactide) based fibrous scaffolds for ligament tissue engineering

The aim of this study was to fabricate a fibrous scaffold that closely resembled the micro-structural architecture and mechanical properties of collagen fibres found in the anterior cruciate ligament (ACL). To achieve this aim, fibrous scaffolds were made by electrospinning L-lactide based polymers. L-Lactide was chosen primarily due to its demonstrated biocompatibility, biodegradability and high modulus. The electrospun fibres were collected in tension on a rotating wire mandrel. Upon treating these fibres in a heated aqueous environment, they possessed a crimp-like pattern having a wavelength and amplitude similar to that of native ACL collagen. Of the polymer fibre scaffolds studied, those made from poly(L-lactide-co-D,L-lactide) PLDLA exhibited the highest modulus and were also the most resilient to in vitro hydrolytic degradation, undergoing a slight decrease in modulus compared to the other polymeric fibres over a 6 month period. Bovine fibroblasts seeded on the wavy, crimp-like PLDLA fibres attached, proliferated and deposited extracellular matrix (ECM) molecules on the surface of the fibrous scaffold. In addition, the deposited ECM exhibited bundle formation that resembled the fascicles found in native ACL. These findings demonstrate the importance of replicating the geometric microenvironment in developing effective tissue engineering scaffolds.

Self-Crimping, Biodegradable, Electrospun Polymer Microfibers

Semicrystalline poly(l-lactide-co-ε-caprolactone) (P(LLA-CL)) was used to produce electrospun fibers with diameters on the subcellular scale. P(LLA-CL) was chosen because it is biocompatible and its chemical and physical properties are easily tunable. The use of a rotating wire mandrel as a collection device in the electrospinning process, along with high collection speeds, was used to align electrospun fibers. Upon removal of the fibers from the mandrel, the fibers shrunk in length, producing a crimp pattern characteristic of collagen fibrils in soft connective tissues. The crimping effect was determined to be a result of the residual stresses resident in the fibers due to the fiber alignment process and the difference between the operating temperature (T(op)) and the glass-transition temperature (T(g)) of the polymer. The electrospun fibers could be induced to crimp by adjusting the operating temperature to be greater than that of the polymer glass-transition temperature. Moreover, the crimped fibers exhibited a toe region in their stress-strain profile that is characteristic of collagen present in tendons and ligaments. The crimp pattern was retained during in vitro degradation over 4 weeks. Primary bovine fibroblasts seeded onto these crimped fibers attached, proliferated, and deposited extracellular matrix (ECM) molecules on the surface of the fiber mats. These self-crimping fibers hold great promise for use in tissue engineering scaffolds for connective tissues that require fibers similar in structure to that of crimped collagen fibrils.

The effect of continuous culture on the growth and structure of tissue-engineered cartilage

The use of bioreactors for cartilage tissue engineering has become increasingly important as traditional batch‐fed culture is not optimal for in vitro tissue growth. Most tissue engineering bioreactors rely on convection as the primary means to provide mass transfer; however, convective transport can also impart potentially unwanted and/or uncontrollable mechanical stimuli to the cells resident in the construct. The reliance on diffusive transport may not necessarily be ineffectual as previous studies have observed improved cartilaginous tissue growth when the constructs were cultured in elevated volumes of media. In this study, to approximate an infinite reservoir of media, we investigated the effect of continuous culture on cartilaginous tissue growth in vitro. Isolated bovine articular chondrocytes were seeded in high density, 3D culture on Millicell™ filters. After two weeks of preculture, the constructs were cultivated with or without continuous media flow (5–10 μL/min) for a period of one week. Tissue engineered cartilage constructs grown under continuous media flow significantly accumulated more collagen and proteoglycans (increased by 50–70%). These changes were similar in magnitude to the reported effect of through‐thickness perfusion without the need for large volumetric flow rates (5–10μL/min as opposed to 240–800 μL/min). Additionally, tissues grown in the reactor displayed some evidence of the stratified morphology of native cartilage as well as containing stores of intracellular glycogen. Future studies will investigate the effect of long‐term continuous culture in terms of extracellular matrix accumulation and subsequent changes in mechanical function.

The Effect of Serial Passaging on the Proliferation and Differentiation of Bovine Adipose-Derived Stem Cells

Adipose-derived stem cells (ASCs) represent an excellent cell source for the development of regenerative therapies for a broad variety of tissue disorders. Commonly, in vitro expansion is necessary to obtain sufficient cell populations for research purposes and clinical applications. Although it has been demonstrated that human ASCs can maintain their adipogenic, chondrogenic and osteogenic potential in long-term culture (up to 15 passages), it is not guaranteed that a satisfactory level of differentiation is achievable in later passages. In this study, we investigated the self-renewal and multilineage differentiation capacity of bovine ASCs, isolated from the interdigital fat pad, and explored how serial passaging influences the cells. A proliferation study examined the changes in growth kinetics from passage 1 to 5, and multilineage (adipogenesis, chondrogenesis and osteogenesis) differentiation studies were conducted to compare the potential between passage 2 (P2) and passage 5 (P5). From the proliferation study, a statistically significant change in the doubling time did not appear until P5. In the differentiation study, both P2 and P5 ASCs could be stimulated to undergo multilineage differentiation under specific culturing conditions. However, adipogenic and chondrogenic cultures showed significantly lower levels of differentiation in the P5-induced cultures. In contrast, P5-induced osteogenic cultures had higher alkaline phosphatase enzyme activity than P2-induced cultures, suggesting an increase in the osteogenic response with serial passaging. Overall, bovine ASCs are capable of self-renewal and multilineage differentiation; however, long-term in vitro expansion has a negative effect on adipogenic and chondrogenic differentiation, while potentially favoring osteogenesis.

A crimp-like microarchitecture improves tissue production in fibrous ligament scaffolds in response to mechanical stimuli

The aim of this study was to determine the influence of a crimp-like microarchitecture within electrospun polymer scaffolds on fibroblast extracellular matrix (ECM) production when cultured under dynamic conditions. Electrospun poly(L-lactide-co-D,L-lactide) scaffolds possessing a wave pattern similar to collagen crimp (amplitude: 5 μm and wavelength: 46 μm) were seeded with bovine fibroblasts and mechanically stimulated under dynamic uniaxial tension. The effect of strain amplitude (5%, 10% and 20%) was investigated in a short-term stimulation study. The 10% strain amplitude in the stimulated crimp-like fibre scaffold increased only collagen synthesis, while the 20% strain amplitude increased both collagen and sulphated proteoglycan synthesis compared to stimulated uncrimped (straight) fibre scaffolds and unloaded controls (crimp-like static fibre scaffolds). Alternatively, mechanical stimulation of fibroblasts seeded on uncrimped fibre scaffolds induced significant fibroblast proliferation compared to the stimulated crimp-like fibre scaffolds and no-load controls. Long-term, dynamic mechanical stimulation of fibroblasts seeded on crimp-like fibre scaffolds at 10% strain amplitude resulted in significantly up-regulated collagen accumulation and down-regulated sulphated proteoglycan accumulation. Additionally, the fibroblasts seeded on dynamically stimulated crimp-like fibre scaffolds appeared to form bundles that resembled fascicles, a characteristic hierarchical feature of the native ligament. Our findings demonstrate that fibroblasts seeded on crimp-like fibrous scaffolds respond more favourably (increased ECM synthesis and fascicle formation) to dynamic mechanical loading compared to those grown on scaffolds containing uncrimped (straight) fibres.

Microarchitecture for a three-dimensional wrinkled surface platform.

Dr. M. Li, N. Hakimi, Dr. R. Perez, Prof. S. Waldman, Prof. D. K. Hwang Department of Chemical Engineering Ryerson University 350 Victoria Street , Toronto , Ontario M5B 2K3 , Canada E-mail: dkhwang@ryerson.ca Dr. R. Perez Department of Nanobiomedical Science Dankook University Cheonan 330–714 , South Korea Prof. S. Waldman Li Ka Shing Knowledge Institute St. Michael’s Hospital 30 Bond Street , Toronto , Ontario M5B 1W8 , Canada Prof. J. A. Kozinski Lassonde School of Engineering York University 4700 Keele Street , Toronto , Ontario M3J 1P3 , Canada