Herman S. Cheung

New insight into deformation-dependent hydraulic permeability of gels and cartilage, and dynamic behavior of agarose gels in confined compression

Equilibrium, creep, and dynamic behaviors of agarose gels (2.0-14.8%) in confined compression were investigated in this study. The hydraulic permeabilities of gels were determined by curve-fitting creep data to the biphasic model (J. Biomech. Eng. 102 (1980) 73) and found to be similar in value to those published in the literature (AIChE J. 42 (1996) 1220). A new relationship between intrinsic permeability and volume fraction of water was found for agarose gel, capable of predicting deformation-dependent permeabilities of bovine articular cartilage and 2% agarose gel published in literature. This relationship is accurate for gels and cartilage over a wide range of permeabilities (four orders of magnitude variation). The dynamic stiffness of the gels increases with gel concentration and loading frequency (0.01-1.0Hz). The increase in dynamic stiffness with loading frequency is less pronounced for gels with higher concentrations. The results of this study provide a new insight into deformation-dependent permeability behavior of agarose gel and cartilage, and are important for understanding biological responses of cells to interstitial fluid flow in gel or in cartilage under dynamic mechanical loading.

Distribution of Type I, II, III and V in the Pepsin Solubilized Collagens in Bovine Menisci

The inner one-third (IM) of both lateral and medial menisci resembles hyaline cartilage, both in gross appearance and histological examination, while the outer two-thirds (OM) is fibrocartilaginous in appearance. Collagen was extracted with pepsin, purified with anion and cation exchange column chromatographies and examined by differential salt precipitation, cyanogen bromide-peptide analysis and SDS gel electrophoresis. IM constitutes approximately 10% of the wet weight of whole meniscus, is made up of 70% collagen of which 34% is pepsin soluble. IM is composed of 60% type II and 40% type I collagen. OM is made up of 80% collagen of which 17% is pepsin soluble. The predominant collagen of OM is type I with a trace amount of types III and V (less than 1%).

p53 Down-regulates Human Matrix Metalloproteinase-1 (Collagenase-1) Gene Expression

Recent studies show that the p53 tumor suppressor protein is overexpressed in rheumatoid arthritis (RA) synovium and that somatic mutations previously identified in human tumors are present in RA synovium (Firestein, G. S., Echeverri, F., Yeo, M., Zvaifler, N. J., and Green, D. R. (1997)Proc. Natl. Acad. Sci. U. S. A. 94, 10895–10900; Firestein, G. S., Nguyen, K., Aupperle, K. R., Yeo, M., Boyle, D. L., and Zvaifler, N. J. (1996) Am. J. Pathol. 149, 2143–2151; Reme, T., Travaglio, A., Gueydon, E., Adla, L., Jorgensen, C., and Sany, J. (1998) Clin. Exp. Immunol. 111, 353–3581). We hypothesize that the abnormality of p53 seen in RA synovium may contribute to joint degeneration through the regulation of human matrix metalloproteinase-1 (hMMP-1, collagenase-1) gene expression. Transcription assays were performed with luciferase reporters driven by the promoter of the hMMP-1 gene or by a minimal promoter containing tandem repeats of the consensus binding sequence for activator protein-1, cotransfected with p53-expressing plasmids. The results revealed that (i) wild-type (wt) p53 down-regulated the promoter activity of hMMP-1 in a dose-dependent fashion; (ii) four of six p53 mutants (commonly found in human cancers) lost this repression activity; and (iii) this p53 repression activity was mediated at least in part by the activator protein-1 sites found in the hMMP-1 promoter. These findings were further confirmed by Northern analysis. The down-regulation of hMMP-1 gene expression by endogenous wt-p53 was shown by treatment of U2-OS cells, a wt-p53-containing osteogenic sarcoma line, and Saos-2 cells, a p53-negative osteogenic sarcoma line, with etoposide, a potent inducer of p53 expression. p53, activated by etoposide, appears to block hMMP-1 promoter activity induced by etoposide in U2-OS cells. In summary, we have shown for the first time that the hMMP-1 gene is a p53 target gene, subject to p53 repression. Because MMP-1 is principally responsible for the irreversible destruction of collagen in articular tissue in RA, abnormality of p53 may contribute to joint degeneration through the regulation of MMP-1 expression.

Cyclic compression maintains viability and induces chondrogenesis of human mesenchymal stem cells in fibrin gel scaffolds.

Mechanical loading has long been shown to modulate cartilage-specific extracellular matrix synthesis. With joint motion, cartilage can experience mechanical loading in the form of compressive, tensile or shearing load, and hydrostatic pressure. Recent studies have demonstrated the capacity of unconfined cyclic compression to induce chondrogenic differentiation of human mesenchymal stem cell (hMSC) in agarose culture. However, the use of a nonbiodegradable material such as agarose limits the applicability of these constructs. Of the possible biocompatible materials available for tissue engineering, fibrin is a natural regenerative scaffold, which possesses several desired characteristics including a controllable degradation rate and low immunogenicity. The objective of the present study was to determine the capability of fibrin gels for supporting chondrogenesis of hMSCs under cyclic compression. To optimize the system, three concentrations of fibrin gel (40, 60, and 80 mg/mL) and three different stimulus frequencies (0.1, 0.5, and 1.0 Hz) were used to examine the effects of cyclic compression on viability, proliferation and chondrogenic differentiation of hMSCs. Our results show that cyclic compression (10% strain) at frequencies >0.5 Hz and gel concentration of 40 mg/mL fibrinogen appears to maintain cellular viability within scaffolds. Similarly, variations in gel component concentration and stimulus frequency can be modified such that a significant chondrogenic response can be achieved by hMSC in fibrin constructs after 8 h of compression spread out over 2 days. This study demonstrates the suitability of fibrin gel for supporting the cyclic compression-induced chondrogenesis of mesenchymal stem cells.

Growth of osteoblasts on porous calcium phosphate ceramic: an in vitro model for biocompatibility study.

Biomaterial implantation in animals is commonly used for biocompatibility studies as well as examination of long-term interaction between tissue and the test material. An in vitro cell culture model is proposed as an alternative which will save animal lives and reduce the pain and discomfort of animals used for such studies. In this study the biomaterial was matched to the cell types typical of the implant site of the particular material: porous calcium phosphate ceramic, used as dental and orthopaedic implants, with periosteal fibroblasts, osteoblasts and chondrocytes. All three cell types attached on to the ceramic and formed multicellular layers. Numbers of periosteal fibroblasts, osteoblasts and chondrocytes increased 29-, 23- and 17-fold, respectively, during the 10 wk period. Osteoblasts retained their phenotypic expression by producing only Type I collagen. Parathyroid hormone (PTH, 50 nM) suppressed the alkaline phosphatase activity of osteoblasts by over 50% and increased cAMP by more than 10-fold over control cultures.

Temporal Expression Patterns and Corresponding Protein Inductions of Early Responsive Genes in Rabbit Bone Marrow–Derived Mesenchymal Stem Cells Under Cyclic Compressive Loading

Our recent study suggested that cyclic compressive loading may promote chondrogenesis of rabbit bone‐marrow mesenchymal stem cells (BM‐MSCs) in agarose cultures through the transforming growth factor (TGF)–β signaling pathway. It has been shown that the activating protein 1 (AP‐1) (Jun‐Fos) complex mediated autoinduction of TGF‐β1 and its binding activity was essential for promoting chondrogenesis of mesenchymal cells, whereas Sox9 was identified as an essential transcription factor for chondrogenesis of embryonic mesenchymal cells. The objective of this study was to examine temporal expression patterns of early responsive genes (Sox9, c‐Fos, c‐Jun, and TGF‐β type I and II receptors) and induction of their corresponding proteins in agarose culture of rabbit BM‐MSCs subjected to cyclic compressive loading. The rabbit BM‐MSCs were obtained from the tibias and femurs of New Zealand White rabbits. Cell‐agarose constructs were made by suspending BM‐MSCs in 2% agarose gel (107 cells/ml) for cyclic, unconfined compression tests performed in a custom‐made bioreactor. In the loading experiment, specimens were subjected to sinusoidal loading with a magnitude of 15% strain at a frequency of 1 hertz for 4 hours per day. Experiments were conducted for 2 consecutive days. This study showed that cyclic compressive loading promoted gene expressions of Sox9, c‐Jun, and both TGF‐β receptors and productions of their corresponding proteins, whereas those gene expressions exhibited different temporal expression patterns among genes and between 2 days of testing. The gene expression of c‐Fos was detected only in the samples subjected to1‐hour dynamic compressive loading. These findings suggest that the TGF‐β signal transduction and activities of AP‐1 and Sox9 are involved in the early stage of BM‐MSC chondrogenesis promoted by dynamic compressive loading.

Plasticity of stem cells derived from adult periodontal ligament

BACKGROUND The neural crest contains pluripotent cells that can give rise to neurons and glial cells of the peripheral nervous system, endocrine cells, connective tissue cells, muscle cells and pigment cells during embryonic development. Stem cells derived from the neural crest may still reside in neural crest derivatives including the periodontal ligament (PDL). However, the pluripotency of PDL-derived stem cells has not been investigated. AIM To identify subpopulations of stem cells from the adult PDL and study their pluripotency. Human PDLs were harvested from impacted wisdom teeth (patients aged 19-22 years). RESULTS This study demonstrated that subpopulations of PDL cells expressed embryonic stem cell markers (Oct4, Sox2, Nanog and Klf4) and a subset of neural crest markers (Nestin, Slug, p75 and Sox10). Such PDL cell subpopulations exhibited the potential to differentiate into neurogenic, cardiomyogenic, chondrogenic and osteogenic lineages. Furthermore, preliminary evidence suggesting insulin production of PDL cells might be indicative of the generation of cells of the endodermal lineage. CONCLUSION These findings suggest that the PDL may contain pluripotent stem cells that originate from the neural crest. Our observations open the door to prospective autologous therapeutic applications for a variety of conditions.

Mitogenesis induced by calcium-containing crystals: Role of intracellular dissolution

Hydroxyapatite, like other calcium-containing crystals previously studied by us, is mitogenic for cultured human fibroblasts. This mitogenic effect is not a result of increased ambient calcium concentration due to extracellular crystal dissolution. Synthetic crystals labelled uniformly with calcium 45 (45Ca) undergo endocytosis when incubated with cells and are solubilized. Such solubilization is inhibited by chloroquine or ammonium chloride in concentrations that significantly block the mitogenic effect of crystals but not that of serum. The data suggest that mitogenesis and intracellular crystal dissolution are related phenomena.

Phosphocitrate Inhibits a Basic Calcium Phosphate and Calcium Pyrophosphate Dihydrate Crystal-induced Mitogen-activated Protein Kinase Cascade Signal Transduction Pathway

Calcium deposition diseases caused by calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP) crystals are a significant source of morbidity in the elderly. We have shown previously that both types of crystals can induce mitogenesis, as well as metalloproteinase synthesis and secretion by fibroblasts and chondrocytes. These responses may promote degradation of articular tissues. We have also shown previously that both CPPD and BCP crystals activate expression of the c-fosand c-jun proto-oncogenes. Phosphocitrate (PC) can specifically block mitogenesis and proto-oncogene expression induced by either BCP or CPPD crystals in 3T3 cells and human fibroblasts, suggesting that PC may be an effective therapy for calcium deposition diseases. To understand how PC inhibits BCP and CPPD-mediated cellular effects, we have investigated the mechanism by which BCP and CPPD transduce signals to the nucleus. Here we demonstrate that BCP and CPPD crystals activate a protein kinase signal transduction pathway involving p42 and p44 mitogen-activated protein (MAP) kinases (ERK 2 and ERK 1). BCP and CPPD also cause phosphorylation of a nuclear transcription factor, cyclic AMP response element-binding protein (CREB), on serine 133, a residue essential for CREB’s ability to transactivate. Treatment of cells with PC at concentrations of 10−3 to 10−5 m blocked both the activation of p42/p44 MAP kinases, and CREB serine 133 phosphorylation, in a dose-dependent fashion. At 10−3 m, a PC analogue,n-sulfo-2-aminotricarballylate and citrate also modulate this signal transduction pathway. Inhibition by PC is specific for BCP- and CPPD-mediated signaling, since all three compounds had no effect on serum-induced p42/P44 or interleukin-1β induced p38 MAP kinase activities. Treatment of cells with an inhibitor of MEK1, an upstream activator of MAPKs, significantly inhibited crystal-induced cell proliferation, suggesting that the MAPK pathway is a significant mediator of crystal-induced signals.

In vitro collagen biosynthesis in healing and normal rabbit articular cartilage.

To examine the repair collagens produced by cells in injured cartilage, the femoral articular surfaces of three groups of New Zealand white rabbits were injured by making both superficial and deep lacerations and drill holes. Eight weeks after surgery, the rabbits were killed and slices of injured articular cartilage were harvested. The types of collagen being synthesized at the site of these lesions were identified by labeling the recovered specimens in vitro with 3H-proline and by characterizing the collagen using sodium dodecyl sulphate electrophoresis, carboxymethyl cellulose chromatography, and cyanogen bromide peptide analysis. In all cases, tissue-specific type II ([alpha1 (II)]3) cartilage collagen was synthesized. Histological examination using toluidine blue showed that the chondrocytes bordering the cartilage defect produced by deep lacerations and drill holes responded by increased cellular activity, as shown by cell cloning and increased matrix staining. The drilled holes were completely filled by tissue with staining and morphological characteristics similar to those of hyaline cartilage.