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>
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>