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Biomedical subjects

M M Hyttinen

Publications and source records attributed to M M Hyttinen.

At least 19 recordsLinked to original sources
PubMed2006

Immature porcine knee cartilage lesions show good healing with or without autologous chondrocyte transplantation.

OBJECTIVE: The purpose of this study was to find out how deep chondral lesions heal in growing animals spontaneously and after autologous chondrocyte transplantation. METHODS: A 6mm deep chondral lesion was created in the knee joints of 57 immature pigs and repaired with autologous chondrocyte transplantation covered with periosteum or muscle fascia, with periosteum only, or left untreated. After 3 and 12 months, the repair tissue was evaluated with International Cartilage Repair Society (ICRS) macroscopic grading, modified O'Driscoll histological scoring, and staining for collagen type II and hyaluronan, and with toluidine blue and safranin-O staining for glycosaminoglycans. The repair tissue structure was also examined with quantitative polarized light microscopy and indentation analysis of the cartilage stiffness. RESULTS: The ICRS grading indicated nearly normal repair tissue in 65% (10/17) after the autologous chondrocyte transplantation and 86% (7/8) after no repair at 3 months. At 1 year, the repair tissue was nearly normal in all cases in the spontaneous repair group and in 38% (3/8) in the chondrocyte transplantation group. In most cases, the cartilage repair tissue stained intensely for glycosaminoglycans and collagen type II indicating repair tissue with true constituents of articular cartilage. There was a statistical difference in the total histological scores at 3 months (P=0.028) with the best repair in the spontaneous repair group. A marked subchondral bone reaction, staining with toluidine blue and collagen type II, was seen in 65% of all animals. CONCLUSIONS: The spontaneous repair ability of full thickness cartilage defects of immature pigs is significant and periosteum or autologous chondrocytes do not bring any additional benefits to the repair.

Animals↗
PubMed2005

Quantitative ultrasound imaging of spontaneous repair of porcine cartilage.

OBJECTIVE: Arthroscopy offers qualitative means to evaluate the surface of articular cartilage. However, possible degeneration of the deep cartilage and subchondral bone remains undetected. High frequency ultrasound imaging is an advanced cartilage evaluation method which is conceivable to arthroscopic use and brings diagnostic information also from deeper cartilage and subchondral bone. DESIGN: In this study, we characterized spontaneous repair of porcine cartilage in situ with quantitative 2D-ultrasound imaging. At the age of 7-8 months, a cartilage lesion (diameter 6mm, not penetrating into subchondral bone) was created on the lateral facet of the right femoral trochlea (n=8). The animals were sacrificed 3 months after the surgery. The lesion site, adjacent cartilage and the corresponding control area at the contralateral (left) knee were imaged in situ with 20 MHz ultrasound. Ultrasound reflection coefficients were determined from the cartilage surface (R) and from the cartilage-bone interface (R(bone)). Microtopography of the articular surface was quantified by calculating ultrasound roughness index (URI) parameter from the ultrasonically determined surface profile. RESULTS: Lesion site was spontaneously filled with visually cartilage-like soft tissue with smooth surface. However, ultrasonic images and histological analyses revealed erosion of subchondral bone under the lesion site. Ultrasound reflection (R) at the surface of the spontaneously repaired tissue was significantly lower (-73.5+/-7.6%, P<0.05) than at the surface of intact cartilage. R(bone) was lowest at the lesion site. The surface roughness of spontaneously repaired cartilage was significantly higher than that of the intact tissue (44.0+/-26.0 microm vs 7.5+/-2.3 microm, P<0.05). CONCLUSIONS: Quantitative ultrasound parameters offered diagnostic information revealing impaired structural integrity of the spontaneously repaired porcine cartilage and subchondral bone. These changes are not detectable by traditional arthroscopic means.

Animals↗
PubMed2005

Abnormal response to physical activity in femurs after heterozygous inactivation of one allele of the Col2a1 gene for type II collagen in mice.

The objective of this study was to evaluate the influence of heterozygous inactivation of one allele of the type II collagen gene (Col2a1) on biomechanical properties and mineral density of bone under physical loading conditions. C57BL/6-TGN mice with heterozygous knockout (HZK) inactivation of Col2a1 gene and their nontransgenic littermate controls were housed in individual cages with running wheels for 9 and 15 months. The running activity of each mouse was monitored continuously throughout the experiment. Bone mineral density (BMD) of mice femora was measured using dual-energy X-ray absorptiometry (DXA) and peripheral quantitative computerized tomography (pQCT). Biomechanical properties were determined using three-point bending tests. Vertebral bone samples were prepared for quantitative polarized light microscopy and digital densitometry of proteoglycans. The concentration of total collagen and collagen cross-links were analyzed using high-performance liquid chromatograpy (HPLC). The average daily running distance was shorter for the HZK mice between the age of 4 and 15 months as compared with normal runners (P < 0.05). The ultimate breaking force was 14.8% and 23.6% (9 vs. 15 months) lower in HZK-runners than in wild-type runners. BMD of the femur was 6.1% lower in HZK-runners at the age of 9 months (P < 0.05). Physical activity increased cortical BMD in wild-type runners but not in the HZK runners at the age of 9 months. The collagen network of the HZK mice was less organized. There were only minor changes in BMD and mechanical and structural properties between sedentary HZK mice and their wild-type controls. Increased physical activity induced significantly lower bone density, mechanical properties, and organization of collagen fibers in male HZK mice. However, there were no major differences in biomechanical parameters between sedentary HZK and wild-type male mice. This suggests an important guiding role of collagen type II in bone remodelling and maturation.

Animals↗
PubMed2003

Subchondral bone reaction associated with chondral defect and attempted cartilage repair in goats.

Repair of cartilage damage with autologous chondrocyte transplantation (ACT) has become popular in clinical use during the past few years. Although clinical results have mostly been successful, several unanswered questions remain regarding the biological mechanism of the repair process. The aim of this study was to develop a goat model for ACT. The repair was not successful due to the graft delamination, but we characterize the subchondral changes seen after the procedure. A chondral lesion was created in 14 goat knees, operated on 1 month later with ACT, and covered with periosteum or a bioabsorbable poly-L/D-lactide scaffold. After 3 months, only two of the five lesions repaired with ACT showed partly hyaline-like repair tissue, and all lesions (n = 4) with the scaffold failed. Even though the lesions did not extend through the calcified cartilage, the bone volume and collagen organization of bone structure were decreased when assessed by quantitative polarized light microscopy. There was a significant loss of bone matrix and distortion of the trabecular structure of subchondral bone, which extended several millimeters into the bone. The subchondral bone demonstrated strong hyaluronan staining in the bone marrow and cartilaginous areas with signs of endochondral ossification, suggesting structural remodeling of the bone. The goat model used here proved not to be an optimal model for ACT. The changes in subchondral bone may alter the biomechanical properties of the subchondral plate and thus the long-term survival of the repair tissue after ACT.

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PubMed2003

Analysis of lapine cartilage matrix after radiosynovectomy with holmium-166 ferric hydroxide macroaggregate.

OBJECTIVE: To study the short and long term effects of radiosynovectomy on articular cartilage in growing and mature rabbits. METHODS: The articular cartilage of the distal femurs of rabbits was examined four days, two months, and one year after radiosynovectomy with holmium-166 ferric hydroxide macroaggregate ([(166)Ho]FHMA). Arthritic changes were evaluated from histological sections by conventional and polarised light microscopy, and glycosaminoglycan measurements using safranin O staining, digital densitometry, and uronic acid determination. Proteoglycan synthesis was studied by metabolic [(35)]sulphate labelling followed by autoradiography, and electrophoretic analysis of extracted proteoglycans. Northern analyses were performed to determine the mRNA levels of type II collagen, aggrecan, and Sox9 in cartilage samples. RESULTS: Radiosynovectomy had no major effect on the histological appearance of articular cartilage in mature rabbits, whereas more fibrillation was seen in [(166)Ho]FHMA radiosynovectomised knee joints of growing rabbits two months after treatment, but not after one year. Radiosynovectomy did not cause changes in the glycosaminoglycan content of cartilage or in the synthesis or chemical structure of proteoglycans. No radiosynovectomy related changes were seen in the mRNA levels of type II collagen, whereas a transient down regulation of aggrecan and Sox9 mRNA levels was seen in young rabbits two months after [(166)Ho]FHMA radiosynovectomy. CONCLUSIONS: [(166)Ho]FHMA radiosynovectomy caused no obvious chondrocyte damage or osteoarthritic changes in mature rabbits, but in growing rabbits some transient radiation induced effects were seen--for example, mild cartilage fibrillation and down regulation of cartilage-specific genes.

Age Factors↗
PubMed2002

Site-specific immunostaining for type X collagen in noncalcified articular cartilage of canine stifle knee joint.

Type X collagen is a short-chain collagen that is strongly expressed in hypertrophic chondrocytes. In this study, we used an immunohistochemical technique exploiting a prolonged hyaluronidase unmasking of type X collagen epitopes to show that type X collagen is not restricted to calcified cartilage, but is also present in normal canine noncalcified articular cartilage. A 30 degrees valgus angulation procedure of the right tibia was performed in 15 dogs at the age of 3 months, whereas their nonoperated sister dogs served as controls. Samples were collected 7 and 18 months after the surgery and immunostained for type X collagen. The deposition of type X collagen increased during maturation from age 43 weeks to 91 weeks. In the patella, most of the noncalcified cartilage stained for type X collagen, whereas, in the patellar surface of the femur, it was present mainly in the femoral groove close to cartilage surface. In femoral condyles, the staining localized mostly in the superficial cartilage on the lateral and medial sides, but not in the central weight-bearing area. In tibial condyles, type X collagen was often observed close to the cartilage surface in medial parts of the condyles, although staining could also be seen in the deep zone of the cartilage. Staining for type X collagen appeared strongest at sites where the birefringence of polarized light was lowest, suggesting a colocalization of type X collagen with the collagen fibril arcades in the intermediate zone. No significant difference in type X collagen immunostaining was observed in lesion-free articular cartilage between controls and dogs that underwent a 30 degrees valgus osteotomy. In osteoarthritic lesions, however, there was strong immunostaining for both type X collagen and collagenase-induced collagen cleavage products. The presence of type X collagen in the transitional zone of cartilage in the patella, femoropatellar groove, and in tibial cartilage uncovered by menisci suggests that it may involve a modification of collagen fibril arrangement at the site of collagen fibril arcades, perhaps providing additional support to the collagen network.

Animals↗
PubMed2002

Transgenic mouse models for studying the role of cartilage macromolecules in osteoarthritis.

The development of transgenic technology has made possible the generation of targeted gene-mutated mouse lines suitable for use in experimental osteoarthritis (OA) research. Transgenic mice harbouring mutations in cartilage collagen types II and IX develop early-onset OA and are therefore promising models of age-related OA, even though the mice often show signs of chondrodysplasia. Also, mouse lines harbouring other engineered mutations of the extracellular molecules have given rise to early OA. The molecular background of a few spontaneous mutations in mice has also been clarified and the characterization of the OA phenotype is now in progress. These mutations cause severe chondrodysplasia and death in homozygous mice, but the heterozygous offspring develop the early-onset OA phenotype.

Animals↗
PubMed2002

Lifelong voluntary joint loading increases osteoarthritis in mice housing a deletion mutation in type II procollagen gene, and slightly also in non-transgenic mice.

OBJECTIVES: To investigate the effects of voluntary running on the incidence and severity of osteoarthritis (OA) and associated changes in cartilage matrix and subchondral bone in a transgenic Del1 mouse model for OA. METHODS: Del1 mice and their non-transgenic littermate controls were housed from the age of 5-6 weeks to 15 months in individual cages with running wheels. The running activity of each mouse was monitored for the entire 12 month period. Additional Del1 and control mice were housed in individual cages without running wheels. At the end of the experiment the severity of OA was evaluated by light microscopy, and the articular cartilage matrix changes by digital densitometry and quantitative polarised light microscopy. RESULTS: Lifelong voluntary running increased the incidence and severity of OA significantly in Del1 mice (transgenic runners), and slightly also in non-transgenic runners. Severe OA changes increased from 39% in transgenic non-runners to 90% in transgenic runners (p=0.006) in lateral tibial condyles, and from 24% to 80% (p=0.013) in lateral femoral condyles, respectively. The proteoglycan content of articular cartilage was reduced in transgenic runners in comparison with transgenic non-runners (p=0.0167), but a similar effect was not seen in non-transgenic runners compared with non-transgenic non-runners. No attributable differences were seen in the collagen network of articular cartilage or in the subchondral bone between any of the groups. CONCLUSION: The Del1 mutation has earlier been shown to disturb the assembly of the cartilage collagen network and thereby increase the incidence and severity of OA with age. In this study, voluntary running was shown to increase further cartilage damage in the lateral compartments of the knee. This suggests that articular cartilage in Del1 mice is less resistant to physical loading than in control mice. Despite severe OA lesions in the knee joint at the age of 15 months, Del1 mice continued to run voluntarily 2-3 km every night.

Animals↗
PubMed2001

Premature vertebral endplate ossification and mild disc degeneration in mice after inactivation of one allele belonging to the Col2a1 gene for Type II collagen.

STUDY DESIGN: Skeletal tissues of mice with an inactivated allele of the Col2a1 gene for Type II collagen ("heterozygous knockout") were studied. OBJECTIVE: To determine whether a heterozygous inactivation of the Col2a1 gene has a role in the etiology of spine disorders such as disc degeneration. SUMMARY OF BACKGROUND DATA: Mutations in the COL2A1, COL11A1, COL11A2, and COL9A2 genes have been linked to spine disorders. However, the mechanism by which genetic factors lead to disc degeneration still are largely unknown. METHODS: Spine tissues were studied using radiograph analyses; conventional, quantitative, and polarized light microscopy; immunohistochemistry for the major extracellular components, and in situ hybridization for procollagens alpha1(I) and alpha1(II). Voluntary running activity also was monitored in half of the mice. RESULTS: As the findings showed, 1-month-old heterozygous knockout mice had shorter limb bones, skulls, and spines, as well as thicker and more irregular vertebral endplates, which calcified earlier than in the control mice. They also had a lower concentration of glycosaminoglycans in the anulus fibrosus, in the endplates, and in the vertebral bone than the controls. These features in the heterozygous knockout mice were compensated by the age of 15 months. However, the long bones and skulls of the mature heterozygous mice remained shorter than those of the controls. Gene-deficient mice used the running wheel less. However, physical exercise did not induce any marked structural changes in the skeleton. CONCLUSION: Mice with heterozygous knockout of Col2a1 show subtle early skeletal manifestations that bear some resemblance to those of human spine disorders.

Aging↗
PubMed2001

Transgenic mice with inactive alleles for procollagen N-proteinase (ADAMTS-2) develop fragile skin and male sterility.

Transgenic mice were prepared with inactive alleles for procollagen N-proteinase (ADAMTS-2; where ADAMTS stands for a disintegrin and metalloproteinase with thrombospondin repeats). Homozygous mice were grossly normal at birth, but after 1-2 months they developed thin skin that tore after gentle handling. Although the gene was inactivated, a large fraction of the N-propeptides of type I procollagen in skin and the N-propeptides of type II procollagen in cartilage were cleaved. Therefore the results suggested the tissues contained one or more additional enzymes that slowly process the proteins. Electron microscopy did not reveal any defects in the morphology of collagen fibrils in newborn mice. However, in two-month-old mice, the collagen fibrils in skin were seen as bizarre curls in cross-section and the mean diameters of the fibrils were approx. half of the controls. Although a portion of the N-propeptides of type II procollagen in cartilage were not cleaved, no defects in the morphology of the fibrils were seen by electron microscopy or by polarized-light microscopy. Female homozygous mice were fertile, but male mice were sterile with a marked decrease in testicular sperm. Therefore the results indicated that ADAMTS-2 plays an essential role in the maturation of spermatogonia.

Alleles↗
PubMed2001

T2 relaxation reveals spatial collagen architecture in articular cartilage: a comparative quantitative MRI and polarized light microscopic study.

It has been suggested that orientational changes in the collagen network of articular cartilage account for the depthwise T2 anisotropy of MRI through the magic angle effect. To investigate the relationship between laminar T2 appearance and collagen organization (anisotropy), bovine osteochondral plugs (N = 9) were T2 mapped at 9.4T with cartilage surface normal to the static magnetic field. Collagen fibril arrangement of the same samples was studied with polarized light microscopy, a quantitative technique for probing collagen organization by analyzing its ability to rotate plane polarized light, i.e., birefringence (BF). Depthwise variation of safranin O-stained proteoglycans was monitored with digital densitometry. The spatially varying cartilage T2 followed the architectural arrangement of the collagen fibril network: a linear positive correlation between T2 and the reciprocal of BF was established in each sample, with r = 0.91 +/- 0.02 (mean +/- SEM, N = 9). The current results reveal the close connection between the laminar T2 structure and the collagen architecture in histologic zones.

Animals↗
PubMed2001

Age matters: collagen birefringence of superficial articular cartilage is increased in young guinea-pigs but decreased in older animals after identical physiological type of joint loading.

OBJECTIVE: To compare responses of the collagen network and glycosaminoglycans (GAGs) of articular cartilage to physiological type of joint loading in young growing and adult mature guinea-pigs. DESIGN: 10- and 44-week-old guinea-pigs were accustomed to treadmill running for 3 weeks. Thereafter the animals ran 2500 m/day, 5 days a week, for 15 weeks. Articular cartilage specimens from knee joints were collected at 28 and 62 weeks. Osteoarthritis (OA) prevalence and severity was evaluated by aid of light microscopy. The degree of collagen fibril network organization and content was analyzed with quantitative polarized light microscopy. The local concentration of GAGs was determined from cartilage sections with digital densitometry after safranin-O staining. RESULTS: In the young guinea-pigs, running increased up to 24% the optical retardation of polarized light by collagen in the superficial articular cartilage of femur, indicating either a higher degree of fibril assembly and organization or increased amount of collagen, or both. In contrast, in the adult mature animals the optical retardation decreased almost 50% after joint loading (P< 0.01-0.001). Running did not increase cartilage fibrillation. Significant changes in GAG content of cartilage were not found either in the young or adult mature runners. CONCLUSIONS: Increased birefringence of the superficial articular cartilage after joint loading in young guinea-pigs can be interpreted to be a sign of improved and decreased birefringence in older animals a sign of worsened property of the collagen network. It can be suggested therefore that joint loading strengthened the collagen network in the young runners. It can be hypothesized further that with time the inferior property of the collagen network predisposes the older runners to earlier OA than in controls.

Aging↗
PubMed2001

Inactivation of one allele of the type II collagen gene alters the collagen network in murine articular cartilage and makes cartilage softer.

OBJECTIVE: To evaluate the influence of inactivation of one allele ("heterozygous knockout" or "heterozygous inactivation") of the type II procollagen gene (Col2a1) on the biomechanical properties and structure of the articular cartilage and subchondral bone in 15 month old mice. METHODS: Indentation stiffness of the humerus head articular cartilage was measured by a microindentation method. Cartilage and subchondral bone were prepared for digital densitometry of proteoglycans (PGs), polarised light microscopy (PLM) of collagen, and osteoarthrosis (OA) grading. RESULTS: Heterozygous inactivation of the Col2a1 gene softened articular cartilage (p=0.002) as measured by indentation stiffness ((mean (SEM) 0.50 (0.07) MPa v 0.94 (0.13) MPa in controls). Fibrillar collagen network exhibited lower birefringence in the intermediate (p=0.04) and deep zones (p=0.01) of cartilage by PLM, indicating either decreased collagen content or a lower degree of fibril parallelism in the knockout mice. The total and zonal thicknesses of articular cartilage were unchanged. Zonal PG contents did not differ significantly. In knockout mice, the prevalence of superficial fibrillation-that is, a sign of OA, was higher than in controls (73% v 21%, p=0.002). The collagen induced birefringence of the superficial zone was not reduced. The subchondral bone volume fraction was lower in knockout mice than in controls, 31% v 43% (p=0.01), and optical retardation values in PLM of bone collagen were slightly but significantly lower (p=0.01). CONCLUSION: Heterozygous inactivation of the Col2a1 gene made articular cartilage softer, altered the collagenous network, reduced subchondral bone volume, and altered its microstructure. Changes in the cartilage collagen network probably contributed to increased susceptibility to OA.

Animals↗
PubMed2001

Extracellular matrix composition of full-thickness defect repair tissue is little influenced by exercise in rat articular cartilage.

Full-thickness articular cartilage defects in the femoral condyles of adult rats were examined four and eight weeks after injury. Quantitative polarized light microscopic analysis showed that birefringence of the tissue in the central repair area increased more in rats exercised on a treadmill. Glycosaminoglycan content in the repair tissue was also higher than in the intermittent active motion group at four weeks after injury, but by eight weeks the levels were similar in both groups. No normal-looking articular cartilage was formed in the lesions, and only in one animal type II collagen was observed in the superficial zone of repair tissue. No 3B3(-) antigenicity of the proteoglycans was seen during repair. In conclusion, exercise minimally modified the repair of full-thickness articular cartilage defects in adult rats. The repair in the exercised group may occur slightly faster in the early stages but no difference was seen at the eight week time interval between the exercised and the intermittently active group.

Animals↗
PubMed2000

Simultaneous changes in the mechanical properties, quantitative collagen organization, and proteoglycan concentration of articular cartilage following canine meniscectomy.

The mechanical properties and microstructure of articular cartilage from the canine tibial plateau were studied 12 weeks after total medial meniscectomy. The organization of the birefringent collagen network was measured with quantitative polarized light microscopy to determine the thickness and the degree of organization of the superficial and deep zones. The zonal concentration of sulfated glycosaminoglycan was quantified with digital densitometry of safranin-O staining. Equilibrium compressive and shear properties, as well as dynamic shear properties, were measured at sites adjacent to those of microstructural analysis. The results evinced significant loss of cartilage function following meniscectomy, with decreases of 20-50% in the compressive and shear moduli. There was no evidence of alterations in the degree of collagen fibrillar organization, although a complete loss of the surface zone was seen in 60% of the samples that underwent meniscectomy. Meniscectomy resulted in a decreased concentration of sulfated glycosaminoglycan, and significant positive correlations were found between the equilibrium compressive modulus and the glycosaminoglycan content. Furthermore, the shear properties of cartilage correlated directly with collagen fibrillar organization measured at the superficial zone of corresponding sites. These findings demonstrate that meniscectomy leads to impaired mechanical function of articular cartilage, with significant evidence of quantitative correlations between cartilage microstructure and mechanics.

Animals↗
PubMed1999

Biomechanical and structural characteristics of canine femoral and tibial cartilage.

To analyze the interrelationships between the structure, composition, and mechanical properties of articular cartilage, canine knee (n = 10) femoral and tibial cartilages were used as experimental tissues. The biomechanical properties, instant shear modulus (IM), and equilibrium shear modulus (EM) of articular cartilage were investigated using an in situ indentation creep technique. The local variations in the concentration of glycosaminoglycans (GAGs) in the cartilage were measured with a microspectrophotometer after safranin-O staining of histological sections. Using a computer-based quantitative polarized light microscopy method, area-specific measurements of the optical path difference were performed to quantitate collagen-related optical retardation (gamma) of cartilage zones. The IM and EM were 131.3 and 51.2% higher (p < 0.001) in the femoral cartilage than in the tibial cartilage, respectively. The mean thickness of the superficial zone and the relative proportion of the superficial zone from the total uncalcified cartilage was 107.1 and 155.3% higher (p < 0.001) at the femoral test points than in the tibial ones, respectively. The mean thickness of the tibial uncalcified cartilage was 21.1% higher (p < 0.001) than the thickness of the femoral cartilage. The GAG concentration of the tibial cartilage was higher (14.8%, p < 0.001) than that of the femoral cartilage, especially in the superficial zone (50.0%, p < 0.05), whereas the gamma of the collagen network in the superficial zone of the femoral cartilage was 64.7% higher (p < 0.001) than in the tibial cartilage. The percent relative thickness and retardation gamma of the superficial zone correlated positively with the indentation stiffness of the canine knee articular cartilage. These observations indicate that cartilage is structurally inhomogenous and layered tissue and the local organization of collagen and GAG concentration of the articular cartilage regulate the biomechanical properties of the tissue. The structure and composition of the superficial articular cartilage significantly affects the indentation response of the canine knee articular cartilage.

Animals↗