Robin Jacquet

Tissue engineering a model for the human ear: assessment of size, shape, morphology, and gene expression following seeding of different chondrocytes.

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‐ɛ‐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.

Tissue engineering of an auricular cartilage model utilizing cultured chondrocyte-poly(L-lactide-epsilon-caprolactone) scaffolds.

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.

The Nature and Role of Periosteum in Bone and Cartilage Regeneration

This study was undertaken to determine whether periosteum from different bone sources in a donor results in the same formation of bone and cartilage. In this case, periosteum obtained from the cranium and mandible (examples of tissue supporting intramembranous ossification) and the radius and ilium (examples of tissues supporting endochondral ossification) of individual calves was used to produce tissue-engineered constructs that were implanted in nude mice and then retrieved after 10 and 20 weeks. Specimens were compared in terms of their osteogenic and chondrogenic potential by radiography, histology, and gene expression levels. By 10 weeks of implantation and more so by 20 weeks, constructs with cranial periosteum had developed to the greatest extent, followed in order by ilium, radius, and mandible periosteum. All constructs, particularly with cranial tissue although minimally with mandibular periosteum, had mineralized by 10 weeks on radiography and stained for proteoglycans with safranin-O red (cranial tissue most intensely and mandibular tissue least intensely). Gene expression of type I collagen, type II collagen, runx2, and bone sialoprotein (BSP) was detectable on QRT-PCR for all specimens at 10 and 20 weeks. By 20 weeks, the relative gene levels were: type I collagen, ilium >> radial ≧ cranial ≧ mandibular; type II collagen, radial > ilium > cranial ≧ mandibular; runx2, cranial >>> radial > mandibular ≧ ilium; and BSP, ilium ≧ radial > cranial > mandibular. These data demonstrate that the osteogenic and chondrogenic capacity of the various constructs is not identical and depends on the periosteal source regardless of intramembranous or endochondral ossification. Based on these results, cranial and mandibular periosteal tissues appear to enhance bone formation most and least prominently, respectively. The appropriate periosteal choice for bone and cartilage tissue engineering and regeneration should be a function of its immediate application as well as other factors besides growth rate.

Gene expression in slipped capital femoral epiphysis. Evaluation with laser capture microdissection and quantitative reverse transcription-polymerase chain reaction.

BACKGROUND Slipped capital femoral epiphysis is a poorly understood condition affecting adolescents. Prior studies have suggested that the etiology may be related to abnormal collagen in the growth plate cartilage, but we are not aware of any investigations analyzing collagen or other structural proteins on a molecular level in the affected tissue. This study was performed to evaluate expression of mRNA for key structural molecules in growth plate chondrocytes of patients with slipped capital femoral epiphysis. METHODS A core biopsy of the proximal femoral physis was performed in nine patients with slipped capital femoral epiphysis, and the specimens were compared with five specimens from the normal distal femoral and proximal tibial and fibular physes of age-matched patients treated surgically for a limb-length inequality. We utilized laser capture microdissection techniques followed by quantitative reverse transcription-polymerase chain reaction analysis to determine if a change or abnormality in type-II-collagen and/or aggrecan gene expression may be associated with slipped capital femoral epiphysis. With these techniques, we correlated chondrocyte spatial location and gene expression to provide greater insight into this pathological condition and a more complete understanding of growth plate biology in general. RESULTS Downregulation of both type-II collagen and aggrecan was found in the growth plates of the subjects with slipped capital femoral epiphysis when compared with the levels in the age-matched controls. In eight specimens from affected patients, the level of expression of type-II-collagen mRNA was, on the average (and standard error of the mean), 13.7% +/- 0.2% of that in four control specimens and the aggrecan level averaged only 26% +/- 0.2% of the control aggrecan level. CONCLUSIONS The decreases that we identified in type-II-collagen and aggrecan expression would affect the quantity, distribution, and organization of both components in a growth plate, but these changes could be associated with either the cause or the result of a slipped capital femoral epiphysis.

Murine Metapodophalangeal Sesamoid Bones: Morphology and Potential Means of Mineralization Underlying Function

Normal murine metapodophalangeal sesamoid bones, closely associated with tendons, were examined in terms of their structure and mineralization with reference to their potential function following crystal deposition. This study utilized radiography, whole mount staining, histology, and conventional electron microscopy to establish a maturation timeline of mineral formation in 1‐ to 6‐week‐old metapodophalangeal sesamoids from CD‐1 mice. An intimate cellular and structural relationship was documented in more detail than previously described between the sesamoid bone, tendon, and fibrocartilage enthesis at the metapodophalangeal joint. Sesamoid calcification began in 1‐week lateral sesamoids of the murine metacarpophalangeal joint of the second digit. All sesamoids were completely calcified by 4 weeks. Transmission electron microscopy of 2‐week metacarpophalangeal sesamoids revealed extensive Type I collagen in the associated tendon and fibrocartilage insertion sites and Type II collagen and proteoglycan networks in the interior of the sesamoid. No extracellular matrix vesicles were documented. The results demonstrate that murine sesamoid bones consist of cartilage elaborated by chondrocytes that predominantly synthesize and secrete Type II collagen and proteoglycan. Type II collagen and proteoglycans appear responsible for the onset and progression of mineral formation in this tissue. These data contribute to new understanding of the biochemistry, ultrastructure, and mineralization of sesamoids in relation to other bones and calcifying cartilage and tendon of vertebrates. They also reflect on the potentially important but currently uncertain function of sesamoids as serving as a fulcrum point along a tendon, foreshortening its length and altering advantageously its biomechanical properties with respect to tendon‐muscle interaction. Anat Rec, 2010.

Induction of ethanol dependence increases signal peptidase mRNA levels in rat brain.

Differential Northern blot hybridization was used as a screening tool to identify mRNAs that respond quantitatively to the induction of ethanol dependence. Adult male rats were treated with repeated, high doses of ethanol for 4 consecutive days. This regimen resulted in the development of tolerance and dependence upon ethanol. RNA isolated from the ethanol-dependent rat brains was used to construct a cDNA library. One cDNA was identified that hybridized to a mRNA which increased in rat brain during the ethanol treatment. Sequence analysis of the cDNA indicated that it recognized a mRNA in rat brain which was very similar to that which encodes the 18 kDa subunit of canine signal peptidase. The rat signal peptidase mRNA was observed to increase in brain nearly 2-fold within 48 h after the initiation of ethanol treatment. Ethanol did not significantly alter β-actin mRNA levels during the treatment period. These results support the existence of an ethanol-responsive signal peptidase mRNA in rat brain.

Tissue Engineering Models of Human Digits: Effect of Periosteum on Growth Plate Cartilage Development

Tissue-engineered middle phalanx constructs of human digits were investigated to determine whether periosteum wrapped partly about model midshafts mediated cartilage growth plate formation. Models were fabricated by suturing ends of polymer midshafts in a human middle phalanx shape with polymer sheets seeded with heterogeneous chondrocyte populations from bovine articular cartilage. Half of each midshaft length was wrapped with bovine periosteum. Constructs were cultured, implanted in nude mice for up to 20 weeks, harvested and treated histologically to assess morphology and cartilage proteoglycans. After 20 weeks of implantation, chondrocyte-seeded sheets adjacent to periosteum-wrapped midshaft halves established cartilage growth plates resembling normal tissue in vivo. Sheets adjacent to midshafts without periosteum had disorganized cells and no plate formation. Proteoglycans were present at both midshaft ends. Periosteum appears to guide chondrocytes toward growth plate cartilage organization and tissue engineering provides means for carefully examining construct development of this tissue.

Analysis of human osteoarthritic connective tissue by laser capture microdissection and QRT-PCR

Gene expression levels for type II collagen and aggrecan have been determined as potential measures and disease markers of human osteoarthritis in patients undergoing total knee arthroplasty. In this regard, specimens of affected articular cartilage obtained intraoperatively at the time of surgery were placed in RNAlaterTM to maintain RNA integrity and subsequently frozen-sectioned. Individual or small numbers of chondrocytes were isolated by laser capture microdissection and their total RNA was extracted and analyzed by quantitative reverse transcription-polymerase chain reaction. Results indicate that type II collagen and aggrecan mRNA expression from specific cells in osteoarthritic tissues are detectable and reproducible using these approaches. Our work is the first to demonstrate successful isolation of RNA limited to chondrocytes comprising small quantities of human osteoarthritic material. The study presents a new avenue by which the disease and its progression may be critically assayed.

A Method for the Immunohistochemical Identification and Localization of Osterix in Periosteum-Wrapped Constructs for Tissue Engineering of Bone:

A novel immunohistochemistry (IHC) approach has been developed to label and localize osterix, a bone-specific transcription factor, within formalin-fixed, paraffin-embedded, tissue-engineered constructs uniquely containing synthetic polymers and human periosteal tissue. Generally, such specimens consisting in part of polymeric materials and mineral are particularly difficult for IHC identification of proteins. Samples here were fabricated from human periosteum, electrospun poly-l-lactic acid (PLLA) nanofibers, and polycaprolactone/poly-l-lactic acid (PCL/PLLA, 75/25) scaffolds and harvested following 10 weeks of implantation in athymic mice. Heat-induced and protease-induced epitope retrieval methods from selected existing protocols were examined to identify osterix. All such protease-induced techniques were unsuccessful. Heat-induced retrieval gave positive results for osterix immunohistochemical staining in sodium citrate/EDTA/Tween 20 with high heat (120C) and pressure (~30 psi) for 10 min, but the heat and pressure levels resulted in tissue damage and section delamination from slides that limited protocol effectiveness. Heat-induced epitope retrieval led to other osterix-positive staining results achieved with minimal impact on structural integrity of the tissue and polymers in sodium citrate/EDTA/Tween 20 buffer at 60C and normal pressure (14.5 psi) for 72 hr. The latter approach identified osterix-positive cells by IHC within periosteal tissue, layers of electrospun PLLA nanofibers, and underlying PCL/PLLA scaffolds of the tissue-engineered constructs.

Histochemical Analyses of Tissue-Engineered Human Menisci

The field of tissue engineering remains one of the least explored areas of current meniscal research but holds great promise. In this investigation, meniscal fibrochondrocytes were isolated from fresh human meniscal tissue and seeded onto synthetic polyglycolic acid (PGA) scaffolds. Constructs were implanted into the dorsal subcutaneous space of athymic nude mice. Control scaffolds, devoid of meniscal cells, were simultaneously implanted in additional mice. Constructs were harvested over 12 weeks and treated with a variety of histochemical stains to analyze general specimen morphology, cellular viability and proliferation, and collagen secretion. Results indicate that meniscal fibrochondrocyte proliferation increased over the time of implantation with cellular consolidation occurring as the PGA scaffolding was progressively hydrolyzed. Collagen production also increased over time. There were favorable similarities between constructs and human meniscal controls in terms of cellular morphology, phenotypic expression, and collagen production. These initial findings demonstrate procedures supporting proliferation of meniscal fibrochondrocytes, expression of fibrochondral phenotype, and the formation of putative meniscal tissue.