Christopher G. Williams

IN VITRO CHONDROGENESIS OF BONE MARROW-DERIVED MESENCHYMAL STEM CELLS IN A PHOTOPOLYMERIZING HYDROGEL

Mesenchymal stem cells (MSCs) from skeletally mature goats were encapsulated in a photopolymerizing poly(ethylene glycol)-based hydrogel and cultured with or without transforming growth factor beta1 (TGF) to study the potential for chondrogenesis in a hydrogel scaffold system amenable to minimally invasive implantation. Chondrogenic differentiation was evaluated by histological, biochemical, and RNA analyses for the expression of cartilage extracellular matrix components. The two control groups studied were MSCs cultured in monolayer and MSCs encapsulated in the hydrogel and cultured for 6 weeks in chondrogenic medium without TGF-beta1 (6wk-TGF). The three experimental time points for encapsulated cells studied were 0 days (0d), 3 weeks, and 6 weeks in chondrogenic medium with TGF-beta1 at 10 ng/ml (3wk+TGF and 6wk+TGF). MSCs proliferated in the hydrogels with TGF-beta1. Glycosaminoglycan (GAG) and total collagen content of the hydrogels increased to 3.5% dry weight and 5.0% dry weight, respectively, in 6wk+TGF constructs. Immunohistochemistry revealed the presence of aggrecan, link protein, and type II collagen. Upregulation of aggrecan and type II collagen gene expression compared with monolayer MSCs was demonstrated. Type I collagen gene expression decreased from 3 to 6 weeks in the presence of TGF-beta1. 6wk-TGF hydrogels produced no GAG and only moderate amounts of collagen. However, immunohistochemistry and RT-PCR demonstrated a small amount of spontaneous differentiation in this control group. This study demonstrates the ability to encapsulate MSCs to form cartilage-like tissue in vitro in a photopolymerizing hydrogel. This system may be useful for minimally invasive implantation, MSC differentiation, and engineering of composite tissue structures with multiple cellular phenotypes.

Experimental Model for Cartilage Tissue Engineering to Regenerate the Zonal Organization of Articular Cartilage

OBJECTIVE Regeneration of the zonal organization of articular cartilage may be an important advancement for cartilage tissue engineering. The first goal of this study was to validate our surgical technique as a method to selectively isolate chondrocytes from different zones of bovine articular cartilage. The second goal was to confirm that chondrocytes from different zones would have different proliferative and metabolic activities in two-dimensional (2-D) and 3-D cultures. Finally, to regenerate the zonal organization, we sought to make multi-layered constructs by encapsulating chondrocytes from different zones of articular cartilage. DESIGN Cartilage slices were removed from three (upper, middle, and lower) zones of articular cartilage of young bovine legs. Histology and biochemical composition of the cartilage slices were analyzed to confirm that they had been obtained from the proper zone. Growth kinetics and gene expression in monolayer culture and matrix formation in photopolymerizing hydrogels were evaluated. Multi-layered photopolymerizing hydrogels were constructed with chondrocytes from each zone of native cartilage encapsulated. Cell viability and maintenance of the cells in the respective layer were evaluated using the Live/Dead Viability kit and cell tracking protocols, respectively. After 3 weeks, the multi-layered constructs were harvested for histologic examination including immunohistochemistry for type II collagen. RESULTS Analysis of histology and biochemical composition confirmed that the cartilage slices had been obtained from the specific zone. Chondrocytes from different zones differed in growth kinetics and gene expression in monolayer and in matrix synthesis in 3-D culture. Cells encapsulated in each of the three layers of the hydrogel remained viable and remained in the respective layer in which they were encapsulated. After 3-week culture, each zone of multi-layered constructs had similar histologic findings to that of native articular cartilage. CONCLUSION We present this as an experimental model to regenerate zonal organization of articular cartilage by encapsulating chondrocytes from different layers in multi-layered photopolymerizing gels.

Adult stem cell driven genesis of human-shaped articular condyle.

Uniform design of synovial articulations across mammalian species is challenged by their common susceptibility to joint degeneration. The present study was designed to investigate the possibility of creating human-shaped articular condyles by rat bone marrow-derived mesenchymal stem cells (MSCs) encapsulated in a biocompatible poly(ethylene glycol)-based hydrogel. Rat MSCs were harvested, expanded in culture, and treated with either chondrogenic or osteogenic supplements. Rat MSC-derived chondrogenic and osteogenic cells were loaded in hydrogel suspensions in two stratified and yet integrated hydrogel layers that were sequentially photopolymerized in a human condylar mold. Harvested articular condyles from 4-week in vivo implantation demonstrated stratified layers of chondrogenesis and osteogenesis. Parallel in vitro experiments using goat and rat MSCs corroborated in vivo data by demonstrating the expression of chondrogenic and osteogenic markers by biochemical and mRNA analyses. Ex vivo incubated goat MSC-derived chondral constructs contained cartilage-related glycosaminoglycans and collagen. By contrast, goat MSC-derived osteogenic constructs expressed alkaline phosphatase and osteonectin genes, and showed escalating calcium content over time. Rat MSC-derived osteogenic constructs were stiffer than rat MSC-derived chondrogenic constructs upon nanoindentation with atomic force microscopy. These findings may serve as a primitive proof of concept for ultimate tissue-engineered replacement of degenerated articular condyles via a single population of adult mesenchymal stem cells.

In vivo chondrogenesis of mesenchymal stem cells in a photopolymerized hydrogel.

Background: Surgical options for cartilage reconstruction can be significantly improved through advances in cartilage tissue engineering, whereby functional tissue replacements are created by growing cells on polymer scaffolds. The objective of this study was to use a photopolymerizable hydrogel to implant bone marrow–derived mesenchymal stem cells subcutaneously in a minimally invasive manner and promote cartilage tissue formation by the cells in vivo. Methods: Athymic nude mice were injected subcutaneously with polymer solutions of poly(ethylene) oxide diacrylate containing mesenchymal stem cells and placed under a UVA lamp to transdermally photopolymerize (solidify) the injected liquid. Experimental groups included polymer solutions with hyaluronic acid (HA), transforming growth factor (TGF)-β3, or both. After 3 weeks of implantation, cartilage formation was evaluated by gene expression analysis and histologic techniques. Results: Hyaluronic acid increased the viscosity of the polymer solutions, which helped maintain the injections at the desired site during photopolymerization. Mesenchymal stem cells in hydrogels containing both HA and TGF-β3 produced the highest quality cartilage, based on expression of the cartilage-specific genes and production of proteoglycan and collagen II. When used independently, TGF-β3 and HA alone induced cartilage-specific gene expression and collagen type II production; however, TGF-β3 was essential for proteoglycan production. HA enhanced proteoglycan production when combined with TGF-β3 and reduced expression and production of collagen I. Conclusions: This study is the first to demonstrate the minimally invasive implantation and subsequent chondrogenic differentiation of mesenchymal stem cells in the subcutaneous environment. This lays the foundation for further optimization of a novel and practical technology for cartilage reconstruction.

The sympathetic innervation of the human foot.

Background: The sympathetic innervation of the hand was demonstrated using formaldehyde staining techniques in the 1990s and provides a basis for both medical (botulinum toxin type A) and surgical (sympathectomy) therapeutic approaches. This research investigates the sympathetic innervation of the human foot using tyrosine hydroxylase immunohistochemistry. Methods: With institutional review board approval, six freshly amputated lower extremities had arterial, venous, and peripheral nerve biopsies obtained at the distal leg, ankle, and forefoot levels. Tibial, peroneal, sural, and saphenous nerves were processed immediately for immunohistochemical staining using an anti–tyrosine hydroxylase antibody, for light and electron microscopy evaluation. Qualitative assessments noted the presence or absence of tyrosine hydroxylase–positive fibers in artery, vein, and peripheral nerve. Within the nerve, location of the tyrosine hydroxylase staining was noted. Results: The presence of tyrosine hydroxylase–positive material was identified in each artery, vein, and nerve examined at each level of the foot and ankle. For the artery, the staining was in the adventitia, and rarely in the media of the vessel wall. There were clear entry points into the artery from the connective tissue. For the vein, the staining was more evenly distributed but to a lesser intensity than in the artery. Within each nerve at the proximal levels, the staining was diffusely throughout the fascicles, with clear sites of fibers leaving the periphery. Conclusions: It is concluded that (1) sympathetic innervation of the foot arrives along each peripheral nerve, (2) the vessels already contain sympathetic innervation at the level of the ankle, and (3) the sympathetic innervation of the foot is extensive.

An extracellular matrix extract for tissue-engineered cartilage

Cartrigel modulates the chondrogenic effect of TGF-/spl beta/3 on mesenchymal stem cells in photopolymerizing hydrogels. Tissue engineering of cartilage in photopolymerizing PEG-based scaffolds may benefit from the incorporation of ECM extracts for greater control of cell behavior.

A palpable, obstructing carcinoma of the colon incarcerated within a large ventral hernia

Uncovering the etiology of a bowel obstruction in a patient with a hernia represents a diagnostic dilemma. Although the hernia is often initially the presumptive cause of the bowel obstruction, obstructive carcinoma or another pathological process hidden by the hernia are important considerations. Here we describe a case of a man with an obstructing neoplasm of the colon within a large ventral hernia, whose constipation was initially attributed to incarceration of the hernia.