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.

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.

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.

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-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.

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 RNAlater(TM) 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.

Comparison of different chondrocytes for use in tissue engineering of cartilage model structures.

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.

Refinement of collagen-mineral interaction: a possible role for osteocalcin in apatite crystal nucleation, growth and development.

Mineralization of vertebrate tissues such as bone, dentin, cementum, and calcifying tendon involves type I collagen, which has been proposed as a template for calcium and phosphate ion binding and subsequent nucleation of apatite crystals. Type I collagen thereby has been suggested to be responsible for the deposition of apatite mineral without the need for non-collagenous proteins or other extracellular matrix molecules. Based on studies in vitro, non-collagenous proteins, including osteocalcin and bone sialoprotein, are thought to mediate vertebrate mineralization associated with type I collagen. These proteins, as possibly related to mineral deposition, have not been definitively localized in vivo. The present study has reexamined their localization in the leg tendons of avian turkeys, a representative model of vertebrate mineralization. Immunocytochemistry of osteocalcin demonstrates its presence at the surface of, outside and within type I collagen while that of bone sialoprotein appears to be localized at the surface of or outside type I collagen. The association between osteocalcin and type I collagen structure is revealed optimally when calcium ions are added to the antibody solution in the methodology. In this manner, osteocalcin is found specifically located along the a4-1, b1, c2 and d bands defining in part the hole and overlap zones within type I collagen. From these data, while type I collagen itself may be considered a stereochemical guide for intrafibrillar mineral nucleation and subsequent deposition, osteocalcin bound to type I collagen may also possibly mediate nucleation, growth and development of platelet-shaped apatite crystals. Bone sialoprotein and osteocalcin as well, each immunolocalized at the surface of or outside type I collagen, may affect mineral deposition in these portions of the avian tendon.

A study in vivo of the effects of a static compressive load on the proximal tibial physis in rabbits.

BACKGROUND: The effect of compression on the physis is generally defined by the Hueter-Volkmann principle, in which decreased linear growth of the physis results from increased compression. This investigation examined whether mechanically induced compression of rabbit physes causes changes in gene expression, cells, and extracellular components that promote physeal resilience and strength (type-II collagen and aggrecan) and cartilage hypertrophy (type-X collagen and matrix metalloprotease-13). METHODS: Static compressive loads (10 N or 30 N) were applied for two or six weeks across one hind limb proximal tibial physis of thirteen-week-old female New Zealand White rabbits (n = 18). The contralateral hind limb in all rabbits underwent sham surgery with no load to serve as an internal control. Harvested physes were divided into portions for histological, immunohistochemical, and quantitative reverse transcription-polymerase chain reaction analysis. Gene expression was statistically analyzed by means of comparisons between loaded samples and unloaded shams with use of analysis of variance and a Tukey post hoc test. RESULTS: Compared with unloaded shams, physes loaded at 10 N or 30 N for two weeks and at 10 N for six weeks showed histological changes in cells and matrices. Physes loaded at 30 N for six weeks were decreased in thickness and had structurally disorganized chondrocyte columns, a decreased extracellular matrix, and less intense type-II and X collagen immunohistochemical staining. Quantitative reverse transcription-polymerase chain reaction analysis of loaded samples compared with unloaded shams yielded a significantly (p ≤ 0.05) decreased gene expression of aggrecan and type-II and X collagen and no significant (p > 0.05) changes in the matrix metalloprotease-13 gene expression with increasing load. CONCLUSIONS: Compressed rabbit physes generate biochemical changes in collagens, proteoglycan, and cellular and tissue matrix architecture. Changes potentially weaken overall physeal strength, consistent with the Hueter-Volkmann principle, and lend understanding of the causes of pathological conditions of the physis.

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.

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.

Development of bone and cartilage in tissue-engineered human middle phalanx models.

Human middle phalanges were tissue-engineered with midshaft scaffolds of poly(L-lactide-epsilon-caprolactone) [P(LA-CL)], hydroxyapatite-P(LA-CL), or beta-tricalcium phosphate-P(LA-CL) and end plate scaffolds of bovine chondrocyte-seeded polyglycolic acid. Midshafts were either wrapped with bovine periosteum or left uncovered. Constructs implanted in nude mice for up to 20 weeks were examined for cartilage and bone development as well as gene expression and protein secretion, which are important in extracellular matrix (ECM) formation and mineralization. Harvested 10- and 20-week constructs without periosteum maintained end plate cartilage but no growth plate formation. They also consisted of chondrocytes secreting type II collagen and proteoglycan, and they were composed of midshaft regions devoid of bone. In all periosteum-wrapped constructs at like times, end plate scaffolds held chondrocytes elaborating type II collagen and proteoglycan and cartilage growth plates resembling normal tissue. Chondrocyte gene expression of type II collagen, aggrecan, and bone sialoprotein varied depending on midshaft composition, presence of periosteum, and length of implantation time. Periosteum produced additional cells, ECM, and mineral formation within the different midshaft scaffolds. Periosteum thus induces midshaft development and mediates chondrocyte gene expression and growth plate formation in cartilage regions of phalanges. This work is important for understanding developmental principles of tissue-engineered phalanges and by extension those of normal growth plate cartilage and bone.