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Biomedical subjects

Arnold I Caplan

Publications and source records attributed to Arnold I Caplan.

At least 19 recordsLinked to original sources
PubMed2024

Culture Expansion Alters Human Bone Marrow-Derived Mesenchymal Stem Cell Production of Osteoarthritis-Relevant Cytokines and Growth Factors.

PURPOSE: The purposes of this study were to characterize the human bone marrow-derived mesenchymal stem cells (BM-MSCs) production of osteoarthritis-relevant cytokines and growth factors as they are purified and multiplied, a process termed culture expansion, and to compare the immunomodulatory potential of BM-MSCs based on source and medium used for culture expansion. METHODS: BM-MSCs were obtained from iliac crest bone marrow aspirates of 4 healthy donors. These 4 BM-MSC cell lines underwent 4 rounds, or "passages," of the institutional culture expansion protocol, using institutional culture media. The secretory molecules known to play a role in osteoarthritis-related inflammatory immune response, cartilage degradation, and patient symptoms, together called the BM-MSC "secretome," were measured at each passage. Three lines of commercially available BM-MSCs from healthy donors underwent culture expansion by the same protocol, using commercial culture media. The commercial BM-MSCs secretome and the institutional BM-MSCs secretome were compared at each passage. Significance was set at P < .05. RESULTS: Institutional BM-MSCs produced less interleukin-6 at passages 3 (237 &#xb1; 113 pg/mL) and 4 (237 &#xb1; 113 pg/mL) compared with passages 1 (884 &#xb1; 97 pg/mL) and 2 (1071 &#xb1; 129 pg/mL; P < .01). Institutional BM-MSCs produced more macrophage inflammatory protein 3-alpha at passage 4 than at passage 1 (106 &#xb1; 41 vs 32 &#xb1; 7 pg/mL; P < .01). Across passages of culture expansion, institutional BM-MSCs grown on institutional medium expressed more interleukin-6 (P < .001), interleukin-10 (P < .001), interleukin-1 beta (P < .001), tumor necrosis factor alpha (P = .004), and vascular endothelial growth factor C (P = .003) than commercially available BM-MSCs grown on commercial medium. CONCLUSIONS: Culture expansion alters key molecules within the BM-MSC secretome. Additionally, differences in BM-MSC source and culture medium alter the BM-MSC secretome and its immunomodulatory potential. CLINICAL RELEVANCE: This study characterizes the in-vitro changes in BM-MSC secretome during culture expansion based on the cell source and culture medium. It suggests nonequivalence of culture-expanded BM-MSC therapies obtained from different donors using different culture media, even if delivering equivalent numbers of BM-MSCs.

Humans↗
PubMed2006

Clonal characterization of fibroblasts in the superficial layer of the adult human dermis.

The dermis of adult human skin contains a physiologically heterogeneous population of fibroblasts that interact to produce its unique architecture and that participate in inflammatory and wound repair functions in vivo. This heterogeneity has been well documented for fibroblasts located in the superficial papillary dermis and the deep reticular dermis. However, the existence of diverse fibroblast subpopulations within a given region of the dermis has not been explored. In this study, fibroblast cultures have been established from the superficial dermis following enzymatic dissociation of the tissue. These fibroblasts have been cloned by limiting dilution and initially selected on the basis of morphology and proliferation kinetics. Fibroblasts in some of the clones selected for study express alpha-smooth muscle actin, a myofibroblast characteristic. Significant differences for fibroblast clones obtained from the same piece of skin have been observed with regard to their rate of collagen lattice contraction, their ability to organize a fibronectin matrix, their release of specific growth factors/cytokines into culture medium, and their response to interleukin-1alpha. These differences in both morphological and physiological characteristics indicate that the superficial papillary dermis contains a heterogeneous population of fibroblasts. This heterogeneity might indicate that diverse subpopulations of fibroblasts are required to interact in both homeostatic and pathological situations in skin.

Actins↗
PubMed2006

Mesenchymal stem cells as trophic mediators.

Adult marrow-derived Mesenchymal Stem Cells (MSCs) are capable of dividing and their progeny are further capable of differentiating into one of several mesenchymal phenotypes such as osteoblasts, chondrocytes, myocytes, marrow stromal cells, tendon-ligament fibroblasts, and adipocytes. In addition, these MSCs secrete a variety of cytokines and growth factors that have both paracrine and autocrine activities. These secreted bioactive factors suppress the local immune system, inhibit fibrosis (scar formation) and apoptosis, enhance angiogenesis, and stimulate mitosis and differentiation of tissue-intrinsic reparative or stem cells. These effects, which are referred to as trophic effects, are distinct from the direct differentiation of MSCs into repair tissue. Several studies which tested the use of MSCs in models of infarct (injured heart), stroke (brain), or meniscus regeneration models are reviewed within the context of MSC-mediated trophic effects in tissue repair.

Animals↗
PubMed2006

Hyaluronan-based polymer scaffold modulates the expression of inflammatory and degradative factors in mesenchymal stem cells: Involvement of Cd44 and Cd54.

Hyaluronan (HA), in the bone marrow stroma, is the major non-protein glycosaminoglycan component of extracellular matrix (ECM) involved in cell positioning, proliferation, differentiation as well as in receptor-mediated changes in gene expression. Repair of bone and regeneration of bone marrow is dependent on ECM, inflammatory factors, like chemokines and degradative factors, like metalloproteinases. We analyzed the interaction between human mesenchymal stem cells (h-MSCs) and a three-dimensional (3-D) HA-based scaffold in vitro. The expression of CXC chemokines/receptors, CXCL8 (IL-8)/CXCR1-2, CXCL10 (IP-10)/CXCR3, CXCL12 (SDF-1)/CXCR4, and CXCL13 (BCA-1)/CXCR5, and metalloproteinases/inhibitors MMP-1, MMP-3, MMP-13/TIMP-1 were evaluated in h-MSCs grown on plastic or on HA-based scaffold by Real-time PCR, ELISA, and immunocytochemical techniques. Moreover, the expression of two HA receptors, CD44 and CD54, was analyzed. We found both at mRNA and protein levels that HA-based scaffold induced the expression of CXCR4, CXCL13, and MMP-3 and downmodulated the expression of CXCL12, CXCR5, MMP-13, and TIMP-1 while HA-based scaffold induced CD54 expression but not CD44. We found that these two HA receptors were directly involved in the modulation of CXCL12, CXCL13, and CXCR5. This study demonstrates a direct action of a 3-D HA-based scaffold, widely used for cartilage and bone repair, in modulating both h-MSCs inflammatory and degradative factors directly involved in the engraftment of specific cell types in a damaged area. Our data clearly demonstrate that HA in this 3-D conformation acts as a signaling molecule for h-MSCs.

Antibodies, Monoclonal↗
PubMed2006

A rapid seeding technique for the assembly of large cell/scaffold composite constructs.

These studies address critical technical issues involved in creating human mesenchymal stem cell (hMSC)/ scaffold implants for cartilage repair. These issues include obtaining a high cell density and uniform spatial cell distribution within the scaffold, factors that are critical in the initiation and homogeneity of chondrogenic differentiation. For any given scaffold, the initial seeding influences cell density, retention, and spatial distribution within the scaffold, which eventually will affect the function of the construct. Here, we discuss the development of a vacuum-aided seeding technique for HYAFF -11 sponges which we compared to passive infiltration. Our results show that, under the conditions tested, hMSCs were quantitatively and homogeneously loaded into the scaffolds with 90+% retention rates after 24 h in perfusion culture with no negative effect on cell viability or chondrogenic potential. The retention rates of the vacuum-seeded constructs were at least 2 times greater than those of passively seeded constructs at 72 h. Histomorphometric analysis revealed that the core of the vacuum-seeded constructs contained 240% more cells than the core of passively infiltrated scaffolds. The vacuum seeding technique is safe, rapid, reproducible, and results in controlled quantitative cell loading, high retention, and uniform distribution.

Biocompatible Materials↗
PubMed2006

Toxic effects of gentamicin on marrow-derived human mesenchymal stem cells.

We hypothesized that the high concentrations of gentamicin achieved after local administration would have toxic effects on human mesenchymal stem cells. These cells were isolated from bone marrow from three healthy adult donors and cultured with different concentrations of gentamicin (0 microg/mL, 50 microg/mL, 100 microg/mL, and 200 microg/mL) for 7 days. After 7 days of gentamicin exposure, we examined cell viability, proliferation, and in vitro and in vivo osteochondrogenic capacity. Gentamicin did not have an adverse effect on the viability of human mesenchymal stem cells in all test groups, but did inhibit cell proliferation at concentrations of 100 microg/mL and 200 microg/mL. In in vitro osteogenesis, gentamicin decreased the DNA content and alkaline phosphatase activity of human mesenchymal stem cells at an early stage (Days 4 and 8) in a dose-dependent manner. For chondrogenesis, glycosaminoglycan content and Type II and Type X collagen deposition were lower in the pellets made with cells expanded in gentamicin at 100 or 200 microg/mL relative to cells expanded in medium without gentamicin. A comparable effect on osteochondrogenesis was observed in an in vivo model. At a high concentration, gentamicin inhibits proliferation and differentiation of human bone marrow mesenchymal stem cells and could compromise the bone-healing process.

Anti-Bacterial Agents↗
PubMed2005

Sustained Wnt protein expression in chondral constructs from mesenchymal stem cells.

Wnt genes encode a number of secreted glycoproteins which are closely associated with the cell surface and the extracellular matrix. Recently, members of Wnt family have been implicated in regulating chondrocyte differentiation, but their roles in the chondrogenic process are not fully understood. To contribute to an understanding of the roles of Wnts during chondrogenesis, we have analysed the spatiotemporal expression patterns of Wnt using in vitro models for chondrogenesis of human bone marrow-derived mesenchymal stem cells (hMSCs). In chondrogenic aggregate culture system, RT-PCR analysis revealed expression of Wnt5a and Wnt4 during late chondrogenesis (days 10 and 15). Immunohistochemical analysis showed widespread distribution of Wnt5a and Wnt4 throughout the aggregates at this late phase of culture (days 14 and 21). In addition, in this aggregate culture system, immunohistochemical staining of Wnt4 and Wnt5a showed similar spatiotemporal expression patterns to that of type II collagen or type X collagen. To confirm the results obtained by immunostaining, the specificity of the anti-Wnt4 or anti-Wnt5a antibody was assessed by Western blot analysis. Of Wnt4 and Wnt5a, only Wnt5a was immunodetectable by Western blot analysis. Western blot analysis showed that Wnt5a was expressed as two different molecular weight forms of 40 and 44 kDa. Treatment with PNGase F, which removes N-linked oligosaccharides, revealed that the mass difference between these two forms could be accounted for by the N-glycosylation status of the protein. When hMSCs were seeded on a porous gelatin sponge, immunolocalization studies showed that type II collagen and type X collagen were detected particularly at the periphery at day 7 of culture. In contrast, Wnt4 and Wnt5a showed even distribution throughout the hMSC/gelatin sponge constructs. Their different spatial expression patterns suggest that Wnt4 and Wnt5a proteins are not functionally linked to type II collagen and type X collagen synthesis in in vitro chondrogenic models of hMSCs.

Blotting, Western↗
PubMed2005

FGF-2 enhances the mitotic and chondrogenic potentials of human adult bone marrow-derived mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) expanded with and without fibroblast growth factor (FGF) supplementation were compared with respect to their proliferation rate, ability to differentiate along the chondrogenic pathway in vitro, and their gene expression profiles. hMSCs expanded in FGF-supplemented medium were smaller and proliferated more rapidly than hMSCs expanded in control conditions. Chondrogenic cultures made with FGF-treated cells were larger and contain more proteoglycan than those made with control cells. Furthermore, aggregates of FGF-treated cells lacked the collagen type I-positive and collagen type II-negative outer layer characteristic of aggregates of control cells. A total of 358 unique transcripts were differentially expressed in FGF-treated hMSCs. Of these, 150 were upregulated and 208 downregulated. Seventeen percent of these genes affect proliferation. Known genes associated with cellular signaling functions comprised the largest percentage ( approximately 20%) of differentially expressed transcripts. Eighty percent of differentially expressed extracellular matrix-related genes were downregulated. The present findings that FGF-2 enhances proliferation and differentiation of hMSCs adds to a growing body of evidence that cytokines modulate the differentiation potential and, perhaps, the multipotentiality of adult stem cells. With the generation of gene expression profiles of FGF-treated and control cells we have taken the first steps in the elucidation of the molecular mechanism(s) behind these phenomena.

Age Factors↗
PubMed2005

Repair of osteochondral defects with hyaluronan- and polyester-based scaffolds.

OBJECTIVE: The natural repair of osteochondral defects can be enhanced with biocompatible, biodegradable materials that support the repair process. It is our hypothesis that hyaluronan-based scaffolds are superior to synthetic scaffolds because they provide biological cues. We tested this thesis by comparing two hyaluronan-based scaffolds [auto cross-linked polysaccharide polymer (ACP) and HYAFF-11] to polyester-based scaffolds [poly(DL-lactic-co-glycolic acid) (PLGA) and poly(L-lactic acid) (PLLA)] with similar pore size, porosity and degradation times. DESIGN: Fifty-four rabbits received bilateral osteochondral defects. One defect received a hyaluronan-based scaffold and the contralateral defect received the corresponding polyester-based scaffold. Rabbits were euthanized 4, 12 and 20 weeks after surgery and the condyles dissected and processed for histology. RESULTS: Only ACP-treated defects presented bone at the base of the defect at 4 weeks. At 12 weeks, only defects treated with rapidly dissolving implants (ACP and PLGA) presented bone reconstitution consistently, while bone was present in only one third of those treated with slowly dissolving scaffolds (HYAFF-11 and PLLA). After 20 weeks, the articular surface of PLGA-treated defects presented fibrillation more frequently than in ACP-treated defects. The surface of defects treated with slowly dissolving scaffolds presented more cracks and fissures. CONCLUSIONS: The degradation rate of the scaffolds is critical for the repair process. Slowly dissolving scaffolds sustain thicker cartilage at the surface but, it frequently presents cracks and discontinuities. These scaffolds also delay bone formation at the base of the defects. Hyaluronan-based scaffolds appear to allow faster cell infiltration leading to faster tissue formation. The degradation of ACP leads to rapid bone formation while the slow degradation of HYAFF-11 prolongs the presence of cartilage and delays endochondral bone formation.

Animals↗
PubMed2005

A simple method for stem cell labeling with fluorine 18.

Hexadecyl-4-[(18)F]fluorobenzoate ([(18)F]HFB), a long chain fluorinated benzoic acid ester, was prepared in a one-step synthesis by aromatic nucleophilic substitution of [(18)F]fluoride ion on hexadecyl-4-(N,N,N-trimethylammonio)benzoate. The radiolabeled ester was obtained in good yields (52% decay corrected) and high purity (97%). [(18)F]HFB was used to radiolabel rat mesenchymal stem cells (MSCs) by absorption into cell membranes. MicroPET imaging of [(18)F]HFB-labeled MSCs following intravenous injection into the rat showed the expected high and persistent accumulation of radioactivity in the lungs. [(18)F]HFB is thus simple to prepare and uses labeling agent for short-term distribution studies of injected stem cells.

Animals↗
PubMed2004

Controlled release of hyaluronan oligomers from biodegradable polymeric microparticle carriers.

In the present study, biodegradable microparticles of blends of poly(DL-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) were explored as a potential carrier for the controlled release of polysaccharide oligomers. To this end, hyaluronan (HY) oligomers of varying molecular weights were incorporated into PLGA/PEG microparticles. Using a two-level fractional factorial experimental design, four microparticle formulation parameters, the amount of PEG included in the microparticles, the initial HY loading of the microparticles, the molecular weight of HY, and the molecular weight of PLGA, were studied for their influence on the incorporation and in vitro release of HY over the period of 28 days. The entrapment efficiencies were found to range between 10+/-1% and 24+/-2% depending on the initial loading and the molecular weight of the HY oligomer used in the fabrication of the microparticles. The HY was released in a multiphasic fashion including an initial burst release, followed by two separate periods of linear release. The normalized cumulative mass released during the burst release ranged from 25.1+/-9.2% to 93.0+/-0.7% and was found to be significantly influenced by the initial HY loading, the HY molecular weight, and the PLGA molecular weight. The initial period of linear release lasted from day 1 to day 14 and displayed normalized cumulative rates of release from 0.1+/-0.0%/day to 1.4+/-0.2%/day. During this period, PEG content of the microparticles and HY molecular weight exerted the greatest influence on the rate of release. Finally, the second period of linear release lasted through the final time-point at day 28. Here, the normalized cumulative rate of release values ranged from 0.2+/-0.1%/day to 3.6+/-0.7%/day and were dependent on all formulation parameters studied. These results demonstrate the potential of PLGA/PEG blend microparticles for the controlled release of HY oligomers.

Adjuvants, Immunologic↗
PubMed2004

A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential.

Here a new, intrinsically pluripotent, CD45-negative population from human cord blood, termed unrestricted somatic stem cells (USSCs) is described. This rare population grows adherently and can be expanded to 10(15) cells without losing pluripotency. In vitro USSCs showed homogeneous differentiation into osteoblasts, chondroblasts, adipocytes, and hematopoietic and neural cells including astrocytes and neurons that express neurofilament, sodium channel protein, and various neurotransmitter phenotypes. Stereotactic implantation of USSCs into intact adult rat brain revealed that human Tau-positive cells persisted for up to 3 mo and showed migratory activity and a typical neuron-like morphology. In vivo differentiation of USSCs along mesodermal and endodermal pathways was demonstrated in animal models. Bony reconstitution was observed after transplantation of USSC-loaded calcium phosphate cylinders in nude rat femurs. Chondrogenesis occurred after transplanting cell-loaded gelfoam sponges into nude mice. Transplantation of USSCs in a noninjury model, the preimmune fetal sheep, resulted in up to 5% human hematopoietic engraftment. More than 20% albumin-producing human parenchymal hepatic cells with absence of cell fusion and substantial numbers of human cardiomyocytes in both atria and ventricles of the sheep heart were detected many months after USSC transplantation. No tumor formation was observed in any of these animals.

Adipocytes↗
PubMed2004

Fibroblast heterogeneity: more than skin deep.

Dermal fibroblasts are a dynamic and diverse population of cells whose functions in skin in many respects remain unknown. Normal adult human skin contains at least three distinct subpopulations of fibroblasts, which occupy unique niches in the dermis. Fibroblasts from each of these niches exhibit distinctive differences when cultured separately. Specific differences in fibroblast physiology are evident in papillary dermal fibroblasts, which reside in the superficial dermis, and reticular fibroblasts, which reside in the deep dermis. Both of these subpopulations of fibroblasts differ from the fibroblasts that are associated with hair follicles. Fibroblasts engage in fibroblast-epidermal interactions during hair development and in interfollicular regions of skin. They also play an important role in cutaneous wound repair and an ever-increasing role in bioengineering of skin. Bioengineered skin currently performs important roles in providing (1) a basic understanding of skin biology, (2) a vehicle for testing topically applied products and (3) a resource for skin replacement.

Basement Membrane↗
PubMed2004

Targeted delivery of progenitor cells for cartilage repair.

An approach for promoting the adherence of chondrogenic progenitor cells to specific matrix molecules has been tested in a cartilage defect model. Culture-expanded pre-chondrocytes fluorescently labeled with a vital dye were coated by a two-step method wherein lipidated protein G was first allowed to intercalate into cell membranes, and a second incubation in a solution of antibodies to cartilage matrix antigens allowed the binding of the antibodies to the protein G, on the external surface of the cell. The coating technique (termed "cell painting") does not effect cell viability or inhibit growth and chondrogenic potential. Painted cells were then added to rabbit cartilage explants that had a partial-thickness defect, washed, and prepared for histological examination and for confocal microscopy. The histological observations and the confocal observations and fluorescent intensity quantification consistently demonstrated that progenitor cells painted with multiple antibodies were capable of preferential binding to the exposed cartilage matrix within the defect. These results demonstrate that painting cell membranes with antibodies to matrix molecules is an effective method for promoting the adherence of stem or progenitor cells to a cartilage injury site.

Animals↗
PubMed2004

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.

Animals↗
PubMed2004

Osteochondral defect repair by demineralized cortical bone matrix.

It has been reported that demineralized bone matrix (cortical or trabecular bone) contains intrinsic cytokines. In the present study, we tested allogeneic demineralized bone matrix for its capacity to resurface osteochondral defects in a rabbit model with the assumption that the intrinsic cytokines in demineralized bone matrix will facilitate the recruitment of progenitor cells from bone marrow into the defect. It was further assumed that these intrinsic bioactive factors would modulate these cells to differentiate into osteochondrogenic lineage and, thus, functionally repair the osteochondral defect. The biocompatibility of demineralized bone matrix was first tested by loading rabbit bone-marrow-derived mesenchymal stem cells into porous demineralized trabecular bone matrix that was then cultured for 3 days. The cell growth in demineralized trabecular bone matrix was examined by scanning electron microscopy. Loaded rabbit bone-marrow-derived mesenchymal stem cells attached to the trabeculae of demineralized trabecular bone matrix; some cells appeared to be round and others were spread and contacted other cells. Allogeneic rabbit demineralized cortical bone matrix or demineralized trabecular bone matrix was implanted into a full-thickness osteochondral defect in the load-bearing area of the medial femoral condyle of young adult rabbits. At 6 and 12 weeks after surgery, gross and histological examination showed that the defects were repaired up to 95% of their depth. The repair tissue using demineralized cortical bone matrix was composed of subchondral bone and a top layer of cartilage that was smooth and integrated with the adjacent cartilage in most of the specimens. Most of the repair tissue in the defect filled with demineralized trabecular bone matrix had a fibrillated surface without integration with the adjacent cartilage. These results indicate that demineralized cortical bone matrix may be potentially useful to repair osteochondral defects by managing the host's intrinsic reparative cells.

Animals↗