Influence Of Scaffold Composition On Gene Expression And Cellular Organization In Tissue-engineered Middle Phalanx Models Of Human Digits
bone substitutes
To augment or replace defective, diseased, or impaired human digits, the design and development of tissue-engineered phalanges are important and include a middle phalanx model. This construct consists in part of two square-shaped biodegradable polyglycolic acid (PGA) scaffolds (1 x 1 x 0.2 cm in length, width and thickness, respectively) seeded with cartilage cells (chondrocytes) obtained from young calves. One such seeded scaffold is sutured to each end of a rectangular-shaped scaffold (similar to 2 x 0.7 x 0.5 cm in length, width and thickness) serving as the midshaft of the model. To examine the biological regenerative capacity of this biomimetic composite, midshafts were left uncovered or wrapped with periosteum, a tissue from calves giving rise to cartilage and bone. Midshafts were composed of poly(L-lactide-epsilon-caprolactone) CP(LA-CL)] or one of two ceramics, hydroxyapatite (HA) or beta-tricalcium phosphate (beta-TCP), admixed with P(LA-CL). When engineered middle phalanx models were implanted and grown for up to 20 weeks under dorsal skin flaps of athymic (nude) mice, resulting constructs varied in their midshaft bone and end plate cartilage composition and structure. Harvested from mice at 20 weeks, all constructs of P(LA-CL) (n = 3), HA-P(LA-CL) (n = 3), or beta-TCPP(LA-CL) (n = 3) without periosteum developed viable end plate cartilage as determined by Safranin-O staining for chondrocyte-secreted proteoglycans, but cells were not organized as in normal growth plate cartilage of human digits. Midshafts remained effectively absent of cells and completely devoid of mineral. Implanted for the same time 20 week period, constructs (n = 3 for each midshaft type) with periosteum each developed viable end plate cartilage having chondrocytes organized into columns resembling normal growth plate cartilage of digits. Midshafts mineralized through the normal process of endochondral ossification. While these features were common to all composites with periosteum, specific differences occurred among them, apparently depending on midshaft copolymer composition. In particular after 10 or 20 weeks of implantation, gene expression of end plate chondrocytes varied in their levels of type II collagen, aggrecan (proteoglycan), or bone sialoprotein, all markers for development of normal cartilage extracellular matrix and mineralization. These results indicate that the composition of midshaft scaffolds comprising middle phalanx models of human digits affects the composition and structure of both midshaft bone and end plate cartilage of constructs. Continuing studies are defining more completely the relationships between structure and composition of bone and cartilage tissues developed and properties of their underlying copolymer scaffolds in these biomineralized models.
Landis W J; Wada Y; Enjo M; Jacquet R; Lowder E; Isogai N
Structure-Property Relationships in Biomineralized and Biomimetic Composites
2009
2009
Book Chapter
n/a
Tissue Engineering A Model For The Human Ear: Assessment Of Size, Shape, Morphology, And Gene Expression Following Seeding Of Different Chondrocytes
auricular reconstruction; bone sialoprotein; cartilage tissue; Cell Biology; cells; costal cartilage; culture; Dermatology; growth-plate; in-vitro; regeneration; Research & Experimental Medicine; Surgery; transplantation
This study examines the tissue engineering of a human ear model through use of bovine chondrocytes isolated from four different cartilaginous sites (nasoseptal, articular, costal, and auricular) and seeded onto biodegradable poly(l-lactic acid) and poly(l-lactide-epsilon-caprolactone) (50 : 50) polymer ear-shaped scaffolds. After implantation in athymic mice for up to 40 weeks, cell/scaffold constructs were harvested and analyzed in terms of size, shape, histology, and gene expression. Gross morphology revealed that all the tissue-engineered cartilages retained the initial human auricular shape through 40 weeks of implantation. Scaffolds alone lost significant size and shape over the same period. Quantitative reverse transcription-polymerase chain reaction demonstrated that the engineered chondrocyte/scaffolds yielded unique expression patterns for type II collagen, aggrecan, and bone sialoprotein mRNA. Histological analysis showed type II collagen and proteoglycan to be the predominant extracellular matrix components of the various constructs sampled at different implantation times. Elastin was also present but it was found only in constructs seeded with auricular chondrocytes. By 40 weeks of implantation, tissue-engineered cartilage of costal origin became calcified, marked by a notably high relative gene expression level of bone sialoprotein and the presence of rigid, nodular protrusions formed by mineralizing rudimentary cartilaginous growth plates. The collective data suggest that nasoseptal, articular, and auricular cartilages represent harvest sites suitable for development of tissue-engineered human ear models with retention over time of three-dimensional construct architecture, gene expression, and extracellular matrix composition comparable to normal, nonmineralizing cartilages. Calcification of constructs of costal chondrocyte origin clearly shows that chondrocytes from different tissue sources are not identical and retain distinct characteristics and that these specific cells are inappropriate for use in engineering a flexible ear model.
Kusuhara H; Isogai N; Enjo M; Otani H; Ikada Y; Jacquet R; Lowder E; Landis W J
Wound Repair and Regeneration
2009
2009-01
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1111/j.1524-475X.2008.00451.x" target="_blank" rel="noreferrer noopener">10.1111/j.1524-475X.2008.00451.x</a>
Comparison Of Different Chondrocytes For Use In Tissue Engineering Of Cartilage Model Structures
articular-cartilage; auricular cartilage; Cell Biology; construct; growth; in-vitro; regeneration; scaffold; shape; stem-cells; vivo
This study compares bovine chondrocytes harvested from four different animal locations-nasoseptal, articular, costal, and auricular-for tissue-engineered cartilage modeling. While the work serves as a preliminary investigation for fabricating a human ear model, the results are important to tissue-engineered cartilage in general. Chondrocytes were cultured and examined to determine relative cell proliferation rates, type II collagen and aggrecan gene expression, and extracellular matrix production. Respective chondrocytes were then seeded onto biodegradable poly(L-lactide-epsilon-caprolactone) disc-shaped scaffolds. Cell-copolymer constructs were cultured and subsequently implanted in the subcutaneous space of athymic mice for up to 20 weeks. Neocartilage development in harvested constructs was assessed by molecular and histological means. Cell culture followed over periods of up to 4 weeks showed chondrocyte proliferation from the tissue sources varied, as did levels of type II collagen and aggrecan gene expression. For both genes, highest expression was found for costal chondrocytes, followed by nasoseptal, articular, and auricular cells. Retrieval of 20-week discs from mice revealed changes in construct dimensions with different chondrocytes. Greatest disc diameter was found for scaffolds seeded with auricular chondrocytes, followed by those with costal, nasoseptal, and articular cells. Greatest disc thickness was measured for scaffolds containing costal chondrocytes, followed by those with nasoseptal, auricular, and articular cells. Retrieved copolymer alone was smallest in diameter and thickness. Only auricular scaffolds developed elastic fibers after 20 weeks of implantation. Type II collagen and aggrecan were detected with differing expression levels on quantitative RT-PCR of discs implanted for 20 weeks. These data demonstrate that bovine chondrocytes obtained from different cartilaginous sites in an animal may elicit distinct responses during their respective development of a tissue-engineered neocartilage. Thus, each chondrocyte type establishes or maintains its particular developmental characteristics, and this observation is critical in the design and elaboration of any tissue-engineered cartilage model.
Isogai N; Kusuhara H; Ikada Y; Ohtani H; Jacquet R; Hillyer J; Lowder E; Landis W J
Tissue Engineering
2006
2006-04
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1089/ten.2006.12.691" target="_blank" rel="noreferrer noopener">10.1089/ten.2006.12.691</a>
Tissue Engineering Of An Auricular Cartilage Model Utilizing Cultured Chondrocyte-poly(l-lactide-epsilon-caprolactone) Scaffolds
articular-cartilage; Cell Biology; cells; chondrocytes; construct; copolymers; laser capture microdissection; morphogenesis; polymer matrices; reconstruction; total; transplantation
To determine the potential development in vivo of tissue-engineered auricular cartilage, chondrocytes from articular cartilage of bovine forelimb joints were seeded on poly(L-lactic acid-epsilon-caprolactone) copolymer scaffolds molded into the shape of a human ear. Copolymer scaffolds alone in the same shape were studied for comparison. Chondrocyte-seeded copolymer constructs and scaffolds alone were each implanted in dorsal skin flaps of athymic mice for up to 40 weeks. Retrieved specimens were examined by histological and molecular techniques. After 10 weeks of implantation, cell-seeded constructs developed cartilage as assessed by toluidine blue and safranin-O red staining; a vascular, perichondrium-like capsule enveloped these constructs; and tissue formation resembled the auricular shape molded originally. Cartilage matrix formation increased, the capsule persisted, and initial auricular configuration was maintained through implantation for 40 weeks. The presence of cartilage production was correlated with RT-PCR analysis, which showed expression of bovine-specific type II collagen and aggrecan mRNA in cell-seeded specimens at 20 and 40 weeks. Copolymer scaffolds monitored only for 40 weeks failed to develop cartilage or a defined capsule and expressed no mRNA. Extensive vascularization led to scaffold erosion, decrease in original size, and loss of contour and shape. These results demonstrate that poly(L-lactic acid-epsilon-caprolactone) copolymer seeded with articular chondrocytes supports development and maintenance of cartilage in a human ear shape over periods to 40 weeks in this implantation model.
Isogai N; Asamura S; Higashi T; Ikada Y; Morita S; Hillyer J; Jacquet R; Landis W J
Tissue Engineering
2004
2004-05
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1089/1076327041348527" target="_blank" rel="noreferrer noopener">10.1089/1076327041348527</a>
Characterization of the cellular origin of a tissue-engineered human phalanx model by in situ hybridization
cells; Cell Biology; expression; endothelial growth-factor; cartilage; joints
Tissue-engineered models of human phalanges have previously been fabricated from a combination of bovine periosteum, cartilage, tendon, and biodegradable polyglycolic acid and poly-L-lactic acid scaffolds. Resulting constructs implanted in athymic mice for more than 40 weeks developed new bone, cartilage, and tendon and became vascularized, but cell types comprising the constructs were unidentified. The origin of cells in middle phalanx models implanted for 20 weeks in nude mice has been studied by in situ hybridization analyzing species-specific gene expression. Oligonucleotide probes homologous to species-specific gene sequences of bovine type 11 and X collagen, aggrecan, bone sialoprotein, biglycan, and osteopontin, and mouse decorin were labeled with S-35 and hybridized to respective serial sections of bovine tissue, mouse tissue, and phalanx constructs. In situ hybridization showed positive message and tissue-specific localization for all bovine-specific probes examined within cartilaginous and midshaft portions of constructs and negative message for the mouse-specific decorin probe. These data show that osteoblasts and chondrocytes comprising constructs are derived exclusively from their original bovine sources over 20 weeks of implantation. Defining the cellular origin of the models lends insight into their biological, chemical, and physical nature and their growth and development. Maintenance of their initial genotype is crucial for future application of the models in augmenting impaired human phalanges and related tissues.
Chubinskaya S; Jacquet R; Isogai N; Asamura S; Landis W J
Tissue Engineering
2004
2004-07
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1089/1076327041887862" target="_blank" rel="noreferrer noopener">10.1089/1076327041887862</a>
Non-destructive studies of tissue-engineered phalanges by magnetic resonance microscopy and X-ray microtomography
bone; bone-mineral density; cartilage; computed-tomography; computed-tomography; Endocrinology & Metabolism; in-vivo; macromolecules; magnetic resonance microscopy; microct; mr; quantitative; relaxation; scaffolds; tissue engineering; trabecular bone; X-ray microtomography
One of the intents of tissue engineering is to fabricate biological materials for the augmentation or replacement of impaired, damaged, or diseased human tissue. In this context, novel models of the human phalanges have been developed recently through suturing of polymer scaffolds supporting osteoblasts, chondrocytes, and tenocytes to mimic bone, cartilage, and tendon, respectively. Characterization of the model constructs has been accomplished previously through histological and biochemical means, both of which are necessarily destructive to the constructs. This report describes the application of two complementary, non-destructive, non-invasive techniques, magnetic resonance microscopy (MRM) and X-ray microtomography (XMT or quantitative computed tomography), to evaluate the spatial and temporal growth and developmental status of tissue elements within tissue-engineered constructs obtained after 10 and 38 weeks of implantation in athymic (nude) mice. These two times represent respective points at which model middle phalanges are comprised principally of organic components while being largely unmineralized and later become increasingly more mineralized. The spatial distribution of mineralized deposits within intact constructs was readily detected by XMT (qCT) and was comparable to low intensity zones observed on MRM hydration maps. Moreover, the MRM-derived hydration values for mineralized zones were inversely correlated with mineral densities measured by XMT. In addition, the MRM method successfully mapped fat deposits, collagenous tissues, and the hydration state of the soft tissue elements comprising the specimens. These results support the application of non-destructive, non-invasive, quantitative MRM and XMT for the evaluation of constituent tissue elements within complex constructs of engineered implants. (c) 2005 Elsevier Inc. All rights reserved.
Potter K; Sweet D E; Anderson P; Davis G R; Isogai N; Asamura S; Kusuhara H; Landis W J
Bone
2006
2006-03
Journal Article
<a href="http://doi.org/10.1016/j.bone.2005.08.025" target="_blank" rel="noreferrer noopener">10.1016/j.bone.2005.08.025</a>
Design and assessment of a tissue-engineered model of human phalanges and a small joint.
*Bioartificial Organs; *Biomimetic Materials; *Finger Joint; *Finger Phalanges; *Tissue Engineering; Animals; Biological; Bone and Bones; Cartilage; Cattle; Humans; Lactic Acid; Mice; Models; Nude; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers; Tendons
OBJECTIVES: To develop models of human phalanges and small joints by suturing different cell-polymer constructs that are then implanted in athymic (nude) mice. DESIGN: Models consisted of bovine periosteum, cartilage, and/or tendon cells seeded onto biodegradable polymer scaffolds of either polyglycolic acid (PGA) or copolymers of PGA and poly-L-lactic acid (PLLA) or poly-epsilon-caprolactone (PCL) and PLLA. Constructs were fabricated to produce a distal phalanx, middle phalanx, or distal interphalangeal joint. SETTING AND SAMPLE POPULATION: Studies of more than 250 harvested implants were conducted at the Northeastern Ohio Universities College of Medicine. EXPERIMENTAL VARIABLE: Polymer scaffold, cell type, and implantation time were examined. OUTCOME MEASURE: Tissue-engineered specimens were characterized by histology, transmission electron microscopy, in situ hybridization, laser capture microdissection and qualitative and quantitative polymerase chain reaction analysis, magnetic resonance microscopy, and X-ray microtomography. RESULTS: Over periods to 60 weeks of implantation, constructs developed through vascularity from host mice; formed new cartilage, bone, and/or tendon; expressed characteristic genes of bovine origin, including type I, II and X collagen, osteopontin, aggrecan, biglycan, and bone sialoprotein; secreted corresponding proteins; responded to applied mechanical stimuli; and maintained shapes of human phalanges with small joints. CONCLUSION: Results give insight into construct processes of tissue regeneration and development and suggest more complete tissue-engineered cartilage, bone, and tendon models. These should have significant future scientific and clinical applications in medicine, including their use in plastic surgery, orthopaedics, craniofacial reconstruction, and teratology.
Landis W J; Jacquet R; Hillyer J; Lowder E; Yanke A; Siperko L; Asamura S; Kusuhara H; Enjo M; Chubinskaya S; Potter K; Isogai N
Orthodontics & craniofacial research
2005
2005-11
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1111/j.1601-6343.2005.00353.x" target="_blank" rel="noreferrer noopener">10.1111/j.1601-6343.2005.00353.x</a>
Experimental use of fibrin glue to induce site-directed osteogenesis from cultured periosteal cells.
*Fibrin Tissue Adhesive; *Osteogenesis; Animals; Bone and Bones/chemistry/cytology/diagnostic imaging; Cattle; Cells; Cultured; Experimental; Implants; Injections; Mice; Non-programmatic; Nude; Osteopontin; Periosteum/*cytology; Radiography; Sialoglycoproteins/analysis
The purpose of this study was to determine whether a combination of fibrin glue and cultured periosteal cells will result in new bone formation at heterotopic sites in nude mice. Growing cells and developing matrices surrounding periosteal explants from the diaphyses of radii of newborn calves were minced and mixed with fibrin glue in a syringe. The cell/matrix-fibrin glue admixture was then injected into the subcutaneous space on the dorsum of athymic nude mice. After 12 weeks of implantation, gross morphology and histologic investigations showed newly formed bone structures in all cell/matrix-fibrin glue admixtures, but none in fibrin glue injected alone and used as control samples. Osteopontin, a protein important in bone development, was identified by a Western blot assay of the cell/matrix-fibrin glue composite. This study supports the feasibility of initiating site-directed formation of bone structures at heterotopic tissue sites by means of injection of cultured periosteal cells and matrix in a fibrin glue carrier.
Isogai N; Landis W J; Mori R; Gotoh Y; Gerstenfeld L C; Upton J; Vacanti J P
Plastic and reconstructive surgery
2000
2000-03
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1097/00006534-200003000-00019" target="_blank" rel="noreferrer noopener">10.1097/00006534-200003000-00019</a>