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Arnold I Caplan

Publications and source records attributed to Arnold I Caplan.

34 records · Page 2Linked to original sources
PubMed2004

Generation of pluripotent stem cells and their differentiation to the chondrocytic phenotype.

It is well documented that adult cartilage has minimal self-repair ability. Current methods for treatment of cartilage injury focus on the relief of pain and inflammation and have met with limited long-term success. In the forefront of new therapeutic approaches, autologous chondrocyte transplantation is still only applied to a very small percentage of the patient population. Our laboratory has focused on cartilage repair using progenitor cells and studied their differentiation into cartilage. Adult mesenchymal stem cells are an attractive candidate as progenitor cells for cartilage repair because of their documented osteogenic and chondrogenic potential, ease of harvest, and ease of expansion in culture; furthermore, their use will obviate the need for harvesting precious healthy cartilage from a patient to obtain autologous chondrocytes for transplantation. However, the need to induce chondrogenic differentiation in the mesenchymal stem cells is superposed on other technical issues associated with cartilage repair; this adds a level of complexity over using mature chondrocytes. This chapter focuses on the methods involved in the isolation of human mesenchymal stem cells and their differentiation along the chondrogenic lineage. Although we have the technology to accomplish chondrogenic differentiation of stem cells, much is still to be learned regarding the regulatory mechanisms controlling the lineage transitions and maturation of the cartilaginous tissue.

Biopsy, Needle↗
PubMed2004

Cellular interactions and bone healing responses to a novel porous tricalcium phosphate bone graft material.

The use of a porous tricalcium phosphate bone void filler (Cellplex TCP, Wright Medical Technology, Inc, Arlington, Tenn) as an alternative to autograft in bone grafting was studied in benchtop, in vitro cell culture, and in vivo preclinical studies. The experimental design included material property quantification, scaffold seeding with mesenchymal stem cells, and implantation in a rabbit segmental defect model. Measured material properties denoted appropriate composition, porosity, and strengths as compared to the literature. Fluid uptake studies and mesenchymal stem cell affinity revealed the scaffold's capabilities as a suitable host for osteoprogenitor cells. In a 1-cm rabbit diaphyseal segmental defect stabilized with an external fixator, tricalcium phosphate was compared to intact autograft, crushed autograft, and open defects. Torsional strengths and stiffnesses of tricalcium phosphate-treated tibia were greater than or equivalent to both intact and crushed autograft controls. Tricalcium phosphate pores exhibited complete bony infiltration histologically. Collectively, the tricalcium phosphate material properties, cell seeding capabilities, and in vivo biological responses give evidence of this implant's functionality as a potential alternative for autograft.

Bone Substitutes↗
PubMed2003

Cytokines and glucocorticoids differentially regulate APN/CD13 and DPPIV/CD26 enzyme activities in cultured human dermal fibroblasts.

Cultured human dermal fibroblasts coexpress two cell surface ectopeptidases, aminopeptidase N (APN/CD13) and dipeptidyl peptidase IV (DPPIV/CD26). These enzymes catalyze the removal of a single amino acid or a dipeptide from the N-termini of oligopeptides, respectively. They are also localized in a differential pattern in normal, non-sun-exposed, adult skin, a finding that supports the supposition that these enzymes might have different functions in the skin, but relatively little is known about their functions in the skin. A better understanding of how the activities of these enzymes are regulated should increase our understanding of their functions in the skin. APN/CD13 was routinely expressed at higher levels on cultured fibroblasts than was DPPIV/CD26. Treatment of cultured fibroblasts with specific factors differentially modulated the activities of these enzymes. APN/CD13 was significantly upregulated by treatment with interleukin-4 (IL-4), interferon gamma (IFNgamma), and the glucocorticoids dexamethasone and hydrocortisone. In contrast, the regulation of DPPIV/CD26 activity was found to be different and more complex. This enzyme was consistently upregulated by IL-1alpha and IL-1beta, but consistently downregulated by glucocorticoids, tumor necrosis factor alpha (TNFalpha) and transforming growth factor beta(1) (TGFbeta(1)). Thus, although these two enzymes are expressed on the same populations of cultured cells, their activities are differentially regulated. This finding, along with their differential distribution in normal skin, suggests that APN/CD13 and DPPIV/CD26 have different functions in the skin.

CD13 Antigens↗
PubMed2003

Age-related changes in the proteoglycans of human skin. Specific cleavage of decorin to yield a major catabolic fragment in adult skin.

Dramatic changes occur in skin as a function of age, including changes in morphology, physiology, and mechanical properties. Changes in extracellular matrix molecules also occur, and these changes likely contribute to the overall age-related changes in the physical properties of skin. The major proteoglycans detected in extracts of human skin are decorin and versican. In addition, adult human skin contains a truncated form of decorin, whereas fetal skin contains virtually undetectable levels of this truncated decorin. Analysis of this molecule, herein referred to as decorunt, indicates that it is a catabolic fragment of decorin rather than a splice variant. With antibody probes to the core protein, decorunt is found to lack the carboxyl-terminal portion of decorin. Further analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry shows that the carboxyl terminus of decorunt is at Phe(170) of decorin. This result indicates that decorunt represents the amino-terminal 43% of the mature decorin molecule. Such a structure is inconsistent with alternative splicing of decorin and suggests that decorunt is a catabolic fragment of decorin. A neoepitope antiserum, anti-VRKVTF, was generated against the carboxyl terminus of decorunt. This antiserum does not recognize intact decorin in any skin proteoglycan sample tested on immunoblots but recognizes every sample of decorunt tested. The results with anti-VRKVTF confirm the identification of the carboxyl terminus of decorunt. Analysis of collagen binding by surface plasmon resonance indicates that the affinity of decorunt for type I collagen is 100-fold less than that of decorin. This observation correlates with the structural analysis of decorunt, in that it lacks regions of decorin previously shown to be important for interaction with type I collagen. The detection of a catabolic fragment of decorin suggests the existence of a specific catabolic pathway for this proteoglycan. Because of the capacity of decorin to influence collagen fibrillogenesis, catabolism of decorin may have important functional implications with respect to the dermal collagen network.

Adult↗
PubMed2003

Production of a monoclonal antibody, DF-5, that identifies cells at the epithelial-mesenchymal interface in normal human skin. APN/CD13 is an epithelial-mesenchymal marker in skin.

Epithelial-mesenchymal interactions play a critical role in skin development and differentiation, and similar interactions may also regulate the day-to-day proliferation and differentiation events of the epidermis that occur in normal adult skin. This study was directed at identifying molecules that are selectively located at the dermal-epidermal junction in normal adult skin as they may be involved in regulating these homeostatic events. To this end, monoclonal antibodies were raised against the crude cell membrane fraction of cultured human dermal fibroblasts. Screening of antibodies that recognized cell surface antigen on cultured human dermal fibroblasts was followed by determining which of these antibodies selectively localized cells at sites of epithelial-mesenchymal interactions. Antibody DF-5 fit these criteria and was further characterized. This antibody was found to recognize the cell surface ectopeptidase aminopeptidase N (APN), a molecule homologous to the cluster differentiation antigen CD13. Antibody DF-5 and anti-CD13 antibodies both identified cells at sites of epithelial-mesenchymal interactions in fetal, neonatal, and adult human skin, and the APN/CD13 enzyme activity was also identified at these sites. A second ectopeptidase, dipeptidyl peptidase IV (DPPIV) or CD26, presented a significantly different immunohistochemical and histochemical pattern in skin samples, confirming the specificity of the APN/CD13 studies. The function of APN/CD13 in skin has yet to be determined. Its invariant localization at sites of epithelial-mesenchymal interactions argues for a role particular to this region. It may play a role in regulating the activity of neuropeptides or other signaling peptides that are released in this region of skin or it may have an as yet undefined role in mediating communication between dermal and epidermal cells.

Adolescent↗
PubMed2003

Embryonic development and the principles of tissue engineering.

Tissue engineering has, historically, used empirical methods to devise reparative strategies for optimizing the repair of skeletal tissue defects. The acquired experience and observations indicate that several aspects of successful repair protocols involve the engineered recapitulation of certain embryonic events. A careful study of the details of embryonic limb formation and subsequent differentiation events into its component skeletal tissues suggests that aspects of these tissue formation events can provide guiding principles for the tissue-engineered regeneration of skeletal tissues in adults. A thesis is developed in reviewing selected aspects of embryonic limb formation whereby one could articulate broad tissue engineering principles that should be followed in order to regenerate portions of excised or damaged skeletal tissues. Central to the regeneration of skeletal tissues is the conversion of progenitor cells and tissue into the desired specialized tissue. For mesenchymal tissues, this requires the conversion of groups of mesenchymal cells with their relatively modest extracellular matrix (ECM) into functional skeletal tissues characterized by a voluminous and specialized ECM. Because of the absence of the complex signalling cascade characteristic of early embryonic events, it is improbable that adult tissue reconstruction strategies can recapitulate distinctive morphologies while forming newly differentiated skeletal tissues. Thus, tissue-engineered regeneration protocols must provide the scaffolds and boundaries to establish the contours and edges of reparative tissues and then must functionally and molecularly integrate this neo-tissue with the surrounding host tissue. Consequently, such scaffolds must provide the reparative cells or their progenitors or the specific attachment or binding sites for endogenous reparative cells. The scaffolds must also provide the signals to start the reparative process, the means and signals to expand the reparative cells, the space for the unique and oriented specialized ECM and, lastly, the capacity to functionally integrate this neo-tissue in a seamless manner with the host tissue. Several tissue-engineering principles based on the details of embryonic events provide guides for the development of scientific logics for new reparative strategies for the regeneration of skeletal tissues.

Animals↗
PubMed2002

Bioreactors mediate the effectiveness of tissue engineering scaffolds.

We hypothesized that the mechanically active environment present in rotating bioreactors mediates the effectiveness of three-dimensional (3D) scaffolds for cartilage tissue engineering. Cartilaginous constructs were engineered by using bovine calf chondrocytes in conjunction with two scaffold materials (SM) (benzylated hyaluronan and polyglycolic acid); three scaffold structures (SS) (sponge, non-woven mesh, and composite woven/non-woven mesh); and two culture systems (CS) (a bioreactor system and petri dishes). Construct size, composition [cells, glycosaminoglycans (GAG), total collagen, and type-specific collagen mRNA expression and protein levels], and mechanical function (compressive modulus) were assessed, and individual and interactive effects of model system parameters (SM, SS, CS, SM*CS and SS*CS) were demonstrated. The CS affected cell seeding (higher yields of more spatially uniform cells were obtained in bioreactor-grown than dish-grown 3-day constructs) and subsequently affected chondrogenesis (higher cell numbers, wet weights, wet weight GAG fractions, and collagen type II levels were obtained in bioreactor-grown than dish-grown 1-month constructs). In bioreactors, mesh-based scaffolds yielded 1-month constructs with lower type I collagen levels and four-fold higher compressive moduli than corresponding sponge-based scaffolds. The data imply that interactions between bioreactors and 3D tissue engineering scaffolds can be utilized to improve the structure, function, and molecular properties of in vitro-generated cartilage.

Animals↗
PubMed2002

Repair of osteochondral defect with tissue-engineered two-phase composite material of injectable calcium phosphate and hyaluronan sponge.

Articular cartilage has limited capacity for repair. In the present study, tissue-engineered two-phase composite material was used for the repair of osteochondral defects in young adult rabbit knee. This composite material is composed of an injectable calcium phosphate (ICP) and a hyaluronan (HA) derivate of either ACP or HYAFF 11 sponge. The osteochondral defect, 3 mm in diameter and 3 mm deep, was created in the weight-bearing region of the medial femoral condyle. The bone portion of the defect was first filled with ICP to a level approximately 1 mm below the articular surface. HA sponge (3 mm in diameter and 1-1.2 mm thick), with or without loading of autologous bone marrow-derived progenitor cells (MPCs), was then inserted into the defect on top of the ICP as it hardened. Animals were allowed free cage activity postoperatively, and killed 4 or 12 weeks (for the HYAFF 11 sponge group) after the surgery. At 4 weeks, histological examination showed that the defect was filled up to 90-100% of its depth. Whitish repair tissue on the top appeared to be integrated with the surrounding articular cartilage. Four distinct zones of repair tissue were identified: a superficial layer, a chondroid tissue layer, an interface between HA sponge and ICP, and the ICP material. Evidence of extensive osteoclastic and osteoblastic activities was observed in the bone tissue surrounding the defect edge and in ICP material. By 12 weeks, the zonal features of the repair tissue became more distinct; chondrocytes were arranged in a columnar array, and a calcified layer of cartilage was formed beneath the chondroid tissue in some specimens. The healing tissue of the HA sponge material loaded with MPCs had higher cellular density and better integration with the surrounding cartilage than HA sponge material not loaded with MPCs. This study suggests that using a two-phase composite graft may hold potential for the repair of osteochondral defects by providing mechanical support that mimicks subchondral bone, while also providing a chondrogenic scaffold for the top cartilage repair.

Animals↗
PubMed2002

Treatment of osteochondral defects with autologous bone marrow in a hyaluronan-based delivery vehicle.

The natural repair of osteochondral defects can be enhanced with biocompatible, biodegradable and bioactive materials that provide structural support and molecular cuing to stimulate repair. Since bone marrow contains osteochondral progenitor cells and bioactive agents, it is hypothesized that the combination of scaffold and bone marrow would be a superior composite material for osteochondral repair. This hypothesis will be tested by comparing the outcome of osteochondral defects filled with a fibronectin-coated hyaluronan-based sponge (ACP) with or without autologous bone marrow. Thirty-three 4-month-old rabbits received 3-mm diameter osteochondral defects that were then filled with ACP loaded or not with autologous bone marrow. Rabbits were sacrificed at 2, 3, 4, 12, and 24 weeks after surgery and the condyles processed for histologic and immunohistochemical evaluation. The defects were graded with a histologic scoring scale. Except for the 3-week specimens, the histologic appearance of the defects was similar in both groups. Four weeks after surgery, the defects were filled with bone with a top layer of cartilage well integrated with the adjacent cartilage. Twelve and 24 weeks after surgery, the defects again showed bone filling. The primary difference between the 4-week samples and the 12- and 24-week samples was that the layer of cartilage that appeared to be thinner than the adjacent cartilage. At each harvest time, the overall histologic scores of the specimens did not reveal statistical differences between the treatment groups. However, as revealed by the results of the 3-week sacrifices, bone marrow loading appeared to accelerate the first stages of the repair process. The fibronectin-coated hyaluronan-based scaffold appears to organize the natural response and facilitate the integration of the neo-cartilage with the adjacent tissue. The fundamental tissue engineering principles derived from this study should provide guidelines for the development of comparable clinical reconstructive therapies.

Animals↗
PubMed2002

The STRO-1+ marrow cell population is multipotential.

Human marrow-derived STRO-1+ cells were tested for their ability to differentiate into multiple mesenchymal phenotypes. STRO-1+ cells were isolated from long-term cultures of human marrow cells by magnetic immunobeads linked to the antibody STRO-1. Stromal layers generated from STRO-1+ cells, when cocultured without exogenous cytokines for several weeks with CD34+/CD38+ and CD34+/CD38low cells, were able to induce a 10- to 20-fold increase in colony-forming units, and to sustain cobblestone area colony-forming cells with a distinct time course for the different hematopoietic precursors. A myofibroblastic and adipocytic phenotype was observed in hematopoiesis-supportive stromal cells, as shown by electron microscopy and staining of lipids with Nile red. The adipocytic potential of STRO-1+ cells was further demonstrated by flow cytometry of Nile red O-stained cells cultured in adipocyte-induced medium. The same population of STRO-1+ cells cultured in osteoblast-inducing medium was shown to have osteogenic potential, as demonstrated in vitro by the formation of mineral (von Kossa staining), and in vivo by the formation of bone tissue within porous calcium phosphate ceramics implanted into athymic mice. This population of STRO-1+ cells was also shown to have chondrogenic potential in aggregate cultures which displayed a morphologic appearance of cartilage and stained positively with antibodies specific to type II and type X collagens. These results show that the subset of marrow cells that express the STRO-1 antigen is capable of differentiating into multiple mesenchymal lineages including hematopoiesis-supportive stromal cells with a vascular smooth muscle-like phenotype, adipocytes, osteoblasts and chondrocytes.

ADP-ribosyl Cyclase↗
PubMed2002

Immunochemical and mechanical characterization of cartilage subtypes in rabbit.

Cartilage is categorized into three general subgroups, hyaline, elastic, and fibrocartilage, based primarily on morphologic criteria and secondarily on collagen (Types I and II) and elastin content. To more precisely define the different cartilage subtypes, rabbit cartilage isolated from joint, nose, auricle, epiglottis, and meniscus was characterized by immunohistochemical (IHC) localization of elastin and of collagen Types I, II, V, VI, and X, by biochemical analysis of total glycosaminoglycan (GAG) content, and by biomechanical indentation assay. Toluidine blue staining and safranin-O staining were used for morphological assessment of the cartilage subtypes. IHC staining of the cartilage samples showed a characteristic pattern of staining for the collagen antibodies that varied in both location and intensity. Auricular cartilage is discriminated from other subtypes by interterritorial elastin staining and no staining for Type VI collagen. Epiglottal cartilage is characterized by positive elastin staining and intense staining for Type VI collagen. The unique pattern for nasal cartilage is intense staining for Type V collagen and collagen X, whereas articular cartilage is negative for elastin (interterritorially) and only weakly positive for collagen Types V and VI. Meniscal cartilage shows the greatest intensity of staining for Type I collagen, weak staining for collagens V and VI, and no staining with antibody to collagen Type X. Matching cartilage samples were categorized by total GAG content, which showed increasing total GAG content from elastic cartilage (auricle, epiglottis) to fibrocartilage (meniscus) to hyaline cartilage (nose, knee joint). Analysis of aggregate modulus showed nasal and auricular cartilage to have the greatest stiffness, epiglottal and meniscal tissue the lowest, and articular cartilage intermediate. This study illustrates the differences and identifies unique characteristics of the different cartilage subtypes in rabbits. The results provide a baseline of data for generating and evaluating engineered repair cartilage tissue synthesized in vitro or for post-implantation analysis.

Animals↗
PubMedDate not supplied

Tissue engineering of autologous cartilage grafts in three-dimensional in vitro macroaggregate culture system.

In the field of tissue engineering, techniques have been described to generate cartilage tissue with isolated chondrocytes and bioresorbable or nonbioresorbable biomaterials serving as three-dimensional cell carriers. In spite of successful cartilage engineering, problems of uneven degradation of biomaterial, and unforeseeable cell-biomaterial interactions remain. This study represents a novel technique to engineer cartilage by an in vitro macroaggregate culture system without the use of biomaterials. Human nasoseptal or auricular chondrocytes were enzymatically isolated and amplified in conventional monolayer culture before the cells were seeded into a cell culture insert with a track-etched membrane and cultured in vitro for 3 weeks. The new cartilage formed within the in vitro macroaggregates was analyzed by histology (toluidine blue, von Kossa-safranin O staining), and immunohistochemistry (collagen types I, II, V, VI, and X and elastin). The total glycosaminoglycan (GAG) content of native and engineered auricular as well as nasal cartilage was assayed colorimetrically in a safranin O assay. The biomechanical properties of engineered cartilage were determined by biphasic indentation assay. After 3 weeks of in vitro culture, nasoseptal and auricular chondrocytes synthesized new cartilage with the typical appearance of hyaline nasal cartilage and elastic auricular cartilage. Immunohistochemical staining of cartilage samples showed a characteristic pattern of staining for collagen antibodies that varied in location and intensity. In all samples, intense staining for cartilage-specific collagen types I, II, and X was observed. By the use of von Kossa-safranin O staining a few positive patches-a possible sign of beginning mineralization within the engineered cartilages-were detected. The unique pattern for nasoseptal cartilage is intense staining for type V collagen, whereas auricular cartilage is only weakly positive for collagen types V and VI. Engineered nasal and auricular macroaggregates were negative for anti-elastin antibody (interterritorially). The measurement of total GAG content demonstrated higher GAG content for reformed nasoseptal cartilage compared with elastic auricular cartilage. However, the total GAG content of engineered macroaggregates was lower than that of native cartilage. In spite of the mechanical stability of the auricular macroaggregates, there was no equilibrium of indentation. The histomorphological and immunohistochemical results demonstrate successful cartilage engineering without the use of biomaterials, and identify characteristics unique to hyaline as well as elastic cartilage. The GAG content of engineered cartilage was lower than in native cartilage and the biomechanical properties were not determinable by indentation assay. This study illustrates a novel in vitro macroaggregate culture system as a promising technique for tissue engineering of cartilage grafts. Further long-term in vitro and in vivo studies must be done before this method can be applied to reconstructive surgery of the nose or auricle.

Cartilage, Articular↗
PubMedDate not supplied

Review: mesenchymal stem cells: cell-based reconstructive therapy in orthopedics.

Adult stem cells provide replacement and repair descendants for normal turnover or injured tissues. These cells have been isolated and expanded in culture, and their use for therapeutic strategies requires technologies not yet perfected. In the 1970s, the embryonic chick limb bud mesenchymal cell culture system provided data on the differentiation of cartilage, bone, and muscle. In the 1980s, we used this limb bud cell system as an assay for the purification of inductive factors in bone. In the 1990s, we used the expertise gained with embryonic mesenchymal progenitor cells in culture to develop the technology for isolating, expanding, and preserving the stem cell capacity of adult bone marrow-derived mesenchymal stem cells (MSCs). The 1990s brought us into the new field of tissue engineering, where we used MSCs with site-specific delivery vehicles to repair cartilage, bone, tendon, marrow stroma, muscle, and other connective tissues. In the beginning of the 21st century, we have made substantial advances: the most important is the development of a cell-coating technology, called painting, that allows us to introduce informational proteins to the outer surface of cells. These paints can serve as targeting addresses to specifically dock MSCs or other reparative cells to unique tissue addresses. The scientific and clinical challenge remains: to perfect cell-based tissue-engineering protocols to utilize the body's own rejuvenation capabilities by managing surgical implantations of scaffolds, bioactive factors, and reparative cells to regenerate damaged or diseased skeletal tissues.

Bioprosthesis↗
PubMedDate not supplied

Effect of transforming growth factor beta 2 on marrow-infused foam poly(propylene fumarate) tissue-engineered constructs for the repair of critical-size cranial defects in rabbits.

This study investigates the osseointegration of poly(propylene fumarate) (PPF) with beta-tricalcium phosphate (beta-TCP) scaffolds in a critical-size (diameter, 1.6 cm), cranial defect in 4-month-old rabbits (n = 51), killed at 6 or 12 weeks. Two molecular weights of PPF were used to produce bilayer scaffolds with 0.5-mm solid external and 2.0-mm porous internal layers. The porous layer was infused with bone marrow aspirate, with half the animals receiving 0.8 microg of transforming growth factor beta2 (TGF-beta2). No foreign body or inflammatory response was observed externally or on histological examination of explants. Statistical analysis of histological areal and linear measures of new bone formation found significantly more bone at the later sacrifice time, followed by implants receiving TGF-beta2, followed by low molecular weight PPF implants. Approximately 40% of the explants were tested for incorporation strength with a one-point "push-in" test. Because no permanent fixation was used, implant strength (28.37-129.03 N; range, 6.4 to 29.0 lb of resistance) was due entirely to new bone formation. The strongest bone was seen in implants receiving TGF-beta2-infused marrow in animals killed at 12 weeks. These results support the use of PPF as an osteogenic substrate and future research into preoperative fabrication of critical size and supercritical-size cranial prosthetic implants.

Animals↗