To define the pattern of change at the molecular and cellular levels during the healing of excisional skin wounds in the skeletally immature pig, mRNA levels for relevant molecules were assessed by semiquantitative RT-PCR using porcine specific primer sets and RNA isolated from normal skin and samples at various time post-wounding. Analysis of cellular change was assessed by DNA quantification and histology of tissue sections. The results demonstrated that the changes in the pattern of RNA and DNA content of the scar tissue were consistent with the observed increasing cellularity. The mRNA levels for collagen I, III, HSP47, IL-1, TGF-beta, MMP-1, -2 and -9, TIMP-1, -2, and-4, PAI-1, versican were significantly elevated during healing; levels for biglycan and fibromodulin were not significantly altered; and the mRNA levels for TIMP-3 were depressed. These findings suggest that skin wound healing is a series of complex matrix-cell interactions that involve cellular migration and inflammation, followed by proliferation of fibroblasts with new collagen synthesis, and lastly tissue remodeling of the scar.

Abstract. As limb mesenchymal cells differentiate into chondrocytes, they initiate the synthesis of type II collagen and cease synthesizing type I collagen. Changes in the cytoplasmic levels of type I and type II collagen mRNAs during the course of limb chondrogenesis in vivo and in vitro were examined using cloned cDNA probes. A striking increase in cytoplasmic type II collagen m R N A occurs coincident with the crucial condensation stage of chondrogenesis in vitro, in which prechondrogenic mesenchymal cells become closely juxtaposed before depositing a cartilage matrix. Thereafter, a continuous and progressive increase in the accumulation of cytoplasmic type II coUagen m R N A occurs which parallels the progressive accumulation of cartilage matrix by ceils. The onset of overt chondrogenesis, however, does not involve activation of the transcription of the type II collagen gene. Low levels of type II collagen m R N A are present in the cytoplasm of prechondrogenic mesenchymal cells at the earliest stages of limb development, well before the accumulation of detectable levels of type II collagen. Type I collagen gene expression during chondrogenesis is regulated, at least in part, at the translational level. Type I collagen mRNAs are present in the cytoplasm of differentiated chondrocytes, which have ceased synthesizing detectable amounts of type I collagen. T HE onset of cartilage differentiation in the developinglimb is characterized by a transient cellular condensation or aggregation process in which prechondrogenic mesenchymal cells become closely juxtaposed to one another before initiating cartilage matrix deposition. During this process, a cell-cell interaction, cell shape change, or some other event occurs which is necessary to trigger the chondrogenic differentiation of the cells (15). The critical condensation process may be initiated, at least in part, by a progressive decrease in the accumulation of extracellular hyaluronate (20,35). Fibronectin and type I collagen have been implicated in the cell-cell interaction occurring during condensation (7, 21, 34). Prostaglandin-mediated elevations in cAMP levels during condensation are involved in regulating chondrogenesis (3, 6, 11, 14, 16-18, 32, 33). A change in the shape of the cells from a flattened mesenchymal morphology to a rounded configuration also plays an important role in the process (2, 41).Although considerable insight has been gained into the extracellular influences and intracellular regulatory molecules that are involved in regulating limb chondrogenesis, virtually nothing is known about the molecular mechanisms by which these factors directly influence the changes in gene activity that occur during cartilage differentiation. Prechondrogenic limb mesenchymal cells synthesize type I collagen (36). As the cells differentiate into chondrocytes, they initiate the synthesis of cartilage-characteristic type II collagen and cease synthesizing type I collagen (36). Thus the conversion of mesenchymal chondrogenic progenitor cells into chondrocytes i...

Damage to articular cartilage can eventually lead to osteoarthritis (OA), a debilitating, degenerative joint disease that affects millions of people around the world. The limited natural healing ability of cartilage and the limitations of currently available therapies make treatment of cartilage defects a challenging clinical issue. Hopes have been raised for the repair of articular cartilage with the help of supportive structures, called scaffolds, created through tissue engineering (TE). Over the past two decades, different designs and fabrication techniques have been investigated for developing TE scaffolds suitable for the construction of transplantable artificial cartilage tissue substitutes. Advances in fabrication technologies now enable the strategic design of scaffolds with complex, biomimetic structures and properties. In particular, scaffolds with hybrid and/or biomimetic zonal designs have recently been developed for cartilage tissue engineering applications. This paper reviews critical aspects of the design of engineered scaffolds for articular cartilage repair as well as the available advanced fabrication techniques. In addition, recent studies on the design of hybrid and zonal scaffolds for use in cartilage tissue repair are highlighted.

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Three-dimensional (3D) bioprinting of hybrid constructs is a promising biofabrication method for cartilage tissue engineering because a synthetic polymer framework and cell-impregnated hydrogel provide structural and biological features of cartilage, respectively. During bioprinting, impregnated cells may be subjected to high temperatures (caused by the adjacent melted polymer) and process-induced mechanical forces, potentially compromising cell function. This study addresses these biofabrication issues, evaluating the heat distribution of printed polycaprolactone (PCL) strands and the rheological property and structural stability of alginate hydrogels at various temperatures and concentrations. The biocompatibility of parameters from these studies was tested by culturing 3D hybrid constructs bioprinted with primary cells from embryonic chick cartilage. During initial two-dimensional culture expansion of these primary cells, two morphologically and molecularly distinct cell populations ("rounded" and "fibroblastic") were isolated. The biological performance of each population was evaluated in 3D hybrid constructs separately. The cell viability, proliferation, and cartilage differentiation were observed at high levels in hybrid constructs of both cell populations, confirming the validity of these 3D bioprinting parameters for effective cartilage tissue engineering. Statistically significant performance variations were observed, however, between the rounded and fibroblastic cell populations. Molecular and morphological data support the notion that such performance differences may be attributed to the relative differentiation state of rounded versus fibroblastic cells (i.e., differentiated chondrocytes vs. chondroprogenitors, respectively), which is a relevant issue for cell-based tissue engineering strategies. Taken together, our study demonstrates that bioprinting 3D hybrid constructs of PCL and cell-impregnated alginate hydrogel is a promising approach for cartilage tissue engineering.