Karen E. Yates

Effects of hyaluronan on engineered articular cartilage extracellular matrix gene expression in 3-dimensional collagen scaffolds.

Hyaluronan (HA) is a component of cartilage matrix with known effects on chondrocytes. We tested the effects of adding HA to 3-dimensional (3-D) collagen. sponges on chondrocyte function in vitro. Bovine articular chondrocytes isolated by collagenase digestion were injected into either collagen or HA/collagen scaffolds comprising different amounts of HA (2, 5, 10, and 14% w/w). Expression of aggrecan and type II collagen genes was measured by gene-specific quantitative competitive reverse transcriptase-polymerase chain reactions, and the extracellular matrix was estimated by histomorphometrical analyses. After 7-day culture, the chondrocytes in 2% (w/w) HA sponges expressed fourfold more mRNA transcripts for type II collagen (p = 0.002) and twofold more mRNA transcripts for aggrecan (p = 0.022) than in control collagen sponges. Furthermore, there was 45% more extracellular matrix in 2% (w/w) HA sponges and 43% less matrix in the 10% (w/w) HA sponges compared with plain collagen sponges (p > 0.05). In sum, a small amount of HA in 3-D collagen scaffolds enhanced chondrogenesis, but a greater amount was inhibitory. This 3-D system represents a novel tool to identify mechanisms by which extracellular matrix molecules influence chondrocyte function. Further, these results show the potential for modifying scaffolds to improve production of engineered cartilage for in vivo applications.

Comparative Glycomics of Connective Tissue Glycosaminoglycans

Homeostasis of connective joint tissues depends on the maintenance of an extracellular matrix, consisting of an integrated assembly of collagens, glycoproteins, proteoglycans, and glycosaminoglycans (GAGs). Isomeric chondroitin sulfate (CS) glycoforms differing in position and degree of sulfation and uronic acid epimerization play specific and distinct functional roles during development and disease onset. This work profiles the CS epitopes expressed by different joint tissues as a function of age and osteoarthritis. GAGs were extracted from joint tissues (cartilage, tendon, ligment, muscle, and synovium) and partially depolymerized using chondroitinase enzymes. The oligosaccharide products were differentially stable isotope labeled by reductive amination using 2‐anthranilic acid‐d0 or ‐d4 and subjected to amide‐hydrophilic interaction chromatography (HILIC) online LC‐MS/MS. The analysis presented herein enables simultaneous profiling of the expression of nonreducing end, linker region, and Δ‐unsaturated interior oligosaccharide domains of the CS chains among the different joint tissues. The results provide important new information on the changes to the expression of CS GAG chains during disease and development.

Demineralized bone promotes chondrocyte or osteoblast differentiation of human marrow stromal cells cultured in collagen sponges

Demineralized bone implants have been used for many types of craniomaxillofacial, orthopedic, periodontal, and hand reconstruction procedures. In previous studies, we showed that demineralized bone powder (DBP) induces chondrogenesis of human dermal fibroblasts in a DBP/collagen sponge system that optimized interactions between particles of DBP and target cells in cell culture. In this study, we test the hypothesis that DBP promotes chondrogenesis or osteogenesis of human marrow stromal cells (hMSCs) in 3-D collagen sponge culture, depending upon the culture conditions. We first confirmed that hMSCs have chondrogenic potential when treated with TGF-β, either in 2-D monolayer cultures or in 3-D porous collagen sponges. Second, we found that DBP markedly enhanced chondrogenesis in hMSCs in 3-D sponges, as assessed by metachromasia and expression of chondrocyte-specific genes AGGRECAN, COL II, and COL X. Human dermal fibroblasts (hDFs) were used to define mechanisms of chondroinduction because unlike hMSCs they have no inherent chondrogenic potential. In situ hybridization revealed that hDFs vicinal to DBPs express chondrocyte-specific genes AGGRECAN or COL II. Macroarray analysis showed that DBP activates TGF-β/BMP signaling pathway genes in hDFs. Finally, DBP induced hMSCs to express the osteoblast phenotype when cultured with osteogenic supplements. These studies show how culture conditions can influence the differentiation pathway that human marrow stromal cells follow when stimulated by DBP. These results support the potential to engineer cartilage or bone in vitro by using human bone marrow stromal cells and DBP/collagen scaffolds.

Comparison of TGF-β/BMP Pathways Signaled by Demineralized Bone Powder and BMP-2 in Human Dermal Fibroblasts†

Demineralized bone induces chondrogenic differentiation of human dermal fibroblasts in vitro. Analyses of signaling gene expression showed that DBP and BMP‐2 regulate common and distinct pathways. Although BMP‐2 was originally isolated as a putative active factor in DBP, rhBMP‐2 and DBP do not affect all the same genes or in the same ways.

New Chondrocyte Genes Discovered by Representational Difference Analysis of Chondroinduced Human Fibroblasts

This report includes a review of the potential for gene expression analyses to provide new information for solving problems in skeletal repair and regeneration. It focuses on two approaches: high-throughput gene array methods and representational difference analysis (RDA). The principles underlying these methods are presented with experimental tutorials and some applications. Second, this report includes a review of results from applying both approaches to an in vitro model of postnatal chondroinduction by demineralized bone powder (DBP). Human dermal fibroblasts (hDFs) cultured with DBP acquire a chondroblast phenotype and express cartilage-specific matrix proteins after 7 days. We used cDNA macroarrays and RDA to identify the genes that were altered prior to expression of the chondroblast phenotype, i.e., after only 3 days’ culture with DBP. Using a strategy of data management and reduction based upon biological functions, we reported several functional families of genes (cytoskeletal elements, protein synthesis/trafficking, and matrix molecules and their modifiers) that are upregulated during chondroinduction of hDFs. Together with histological and biochemical evidence of the chondroblast phenotype, the gene expression patterns indicate that there are specific stages of induced chondrocyte differentiation in this experimental system. Third, this report includes a new study, in which DBP-regulated genes were used as a data base to derive new information on the cell biology of chondrocytes. The objective was to determine whether a set of genes expressed during induction of chondrocyte differentiation is also expressed by mature articular chondrocytes. Our search of the literature for 59 of the DBP-regulated genes disclosed that expression of 20 of them (33%) had been documented in mature cartilage or chondrocytes. Of the 39 genes not previously documented in cartilage, 11 were tested by RT-PCR and all were found to be expressed in freshly isolated adult human chondrocytes. This review and these new data show how the strategy of high-throughput methods and functional data reduction can expand our knowledge of chondrocyte cell biology.

Induced chondroblastic differentiation of human fibroblasts by three-dimensional culture with demineralized bone matrix

Demineralized bone powder (DBP) implanted in vivo induces endochondral bone formation (osteoinduction). Connective tissue cells migrate to the powders and they begin to produce cartilage matrix. Subsequently, vascularization is stimulated and the cartilage is replaced by bone and marrow. Demineralized bone implants have been used clinically for a variety of osseous reconstructive applications. We designed a novel three-dimensional (3-D) device to examine the mechanism of chondroinduction in vitro. Normal human dermal fibroblasts (hDFs) were cultured in 3-D collagen sponges with and without DBP. The cells migrated through the collagen lattice and they attached to and spread onto particles of demineralized bone. Cells vicinal to the DBP produced cartilage matrix proteoglycans. Induced cells expressed cartilage-specific gene products, type II collagen and aggrecan. Control hDFs did not produce cartilage matrix when cultured in plain collagen sponges. This experimental system has the potential to reveal mechanisms of gene activation and other early steps in postnatal chondro/osteoinduction. Further, these results suggest that it may be possible to engineer human cartilage for transplantation by culturing autogenous dermal fibroblasts with a chondroinductive agent.

Engineering a Joint: A Chimeric Construct with Bovine Chondrocytes in a Devitalized Chick Knee

This study assessed the feasibility of a devitalized knee as a scaffold for an engineered chimeric joint. Embryonic chick knees (19 days old), devitalized by lyophilization or multiple freeze-thaw cycles, were tested as scaffolds for repopulation with bovine articular chondrocytes (bACs). bACs were seeded into porous three-dimensional collagen sponges and were cultured for 1 day before fabrication of chimeric constructs. A pair of cell-seeded sponges was inserted into the joint space to contact preshaved articular surfaces. In some constructs, a sterile membrane of expanded polytetrafluoroethylene (ePTFE) was inserted between the collagen sponges. Histologic analysis showed that at 1 week, sponges with bACs were adherent to the shaved articular surfaces of the joint with accumulation of metachromatic extracellular matrix. Penetration of bACs and neomatrix into the devitalized matrix appeared to begin in preexistent epiphyseal canals and was observed to some extent in all specimens. Membranes of ePTFE maintained a joint space at 2 and 3 weeks, whereas there was fusion across the two sponges in many specimens lacking the membrane. Gene expression analysis demonstrated that lyophilization, but not multiple freeze-thaw cycles, completely devitalized the chick knees. These studies identified several design parameters crucial for successful engineering of a chimeric joint.

Gene expression changes in an in vitro model of chondroinduction: a comparison of two methods.

There are many useful technologies to describe patterns of gene expression that occur during tissue repair and regeneration. Results from different methods used in one experimental setting are not often compared. In this case study of chondrogenesis, we compare two methods to identify differentially expressed genes, representational difference analysis and targeted macroarray analysis, as a model for investigating genes that may be relevant to tissue repair. We sought to identify genes whose expression was altered when human dermal fibroblasts were cultured in a three‐dimensional, porous collagen sponge with the chondroinductive agent, demineralized bone. Both representational difference analysis and macroarray experiments revealed several functional families of genes as up‐regulated or down‐regulated in chondroinduced fibroblasts. An advantage of representational difference analysis is that altered expression of specific mRNA transcripts can be revealed. In this example, representational difference analysis uncovered the up‐regulation of a specific transcript of Wnt5a in fibroblasts cultured with demineralized bone. Representational difference analysis is limited, however, as there can be false negatives for genes not readily amplified by polymerase chain reaction. We conclude that small arrays containing functional classes of genes can be used to ask specific, hypothesis‐driven questions at minimal cost. It may be prudent, however, to use more than one method to survey differences in gene expression in order to validate and expand findings. (WOUND REP REG 2003;11:386–392)

Engineering a biological joint.

Repair of congenital or acquired joint deformities requires re-creation of compound structures with complex anatomical and functional features. Innovative solutions are needed for many such clinical problems, in early as well as in late stages of degeneration. Progress in musculoskeletal cell, developmental, and molecular biology, advances in in vitro histogenesis, and innovations in materials and manufacturing processes have not yet resulted in widespread clinical applications. An engineered biological joint would enable reconstruction of articular loss resulting from congenital, traumatic, neoplastic, and degenerative conditions. The overall goal of this program is to engineer a biological joint using a devitalized joint from one species as a morphogenic scaffold for chondrocytes from another species and to apply it in a small-animal model. We report the use of a devitalized biological knee as a scaffold for repopulation with chondrocytes. Bovine articular chondrocytes (bACs) were delivered in a porous collagen sponge, previously shown to support chondrogenesis,1 chondroinduction,2 osteogenesis,3 and in vivo compatibility.1,4 Constructs were engineered in vitro and their ectopic in vivo fate was examined. Chimeric joints were made by affixing porous collagen sponges that contained bACs to opposing shaved articular femoral and tibial surfaces of devitalized embryonic (19 day) chick knees. Membranes of expanded polytetrafluoroethylene (ePTFE) were positioned between the femoral and tibial sponges in some constructs (FIG. 1). The constructs were cultured and subsequently transplanted into severe-combined-immunodeficiency-disease (SCID) mice. The constructs were analyzed histologically at intervals in vitro and in vivo and by gene expression analyses for bovine markers. After 1 week in vitro, collagen sponges with bACs were adherent to the shaved articular surfaces of the devitalized chick joints. Initial penetration of neocartilage into the chick scaffold occurred in preexistent canals. Subsequently, metachromatic